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Assay Tips - Abacus ALS

1. If using the same plate keep the plate very clean Alternatively use a second plate for the remaining samples Plate Washing Tips for Reducing Variability Orbital Titer Plate Shaker Lab Line Instruments e Handheld Magnetic Separation Block Model 4625 or equivalent Merck Millipore Cat No 40 285 e Very important For incubating assays overnight e When getting ready to decant the liquid from a power supply must be available for the orbital the plate the plate MUST be firmly attached to shaker in a refrigerator or cold room the magnet The orbital titer plate shaker should be set at Grip the handheld separation block firmly a speed to provide maximum orbital mixing During the wash steps decant with a good without splashing of liquid outside the wells shake while the plate is attached to the magnet For the recommended plate shaker this would over a sink be a setting of 5 7 which is approximately 500 When using a new magnet check for space 800 rpm between the plate and magnet Adjustments However orbital shakers vary Your shaker can require a US Allen hex key to adjust the screws be calibrated by pre wetting the plate with not provided buffer and slowly increasing the speed until splashing occurs Then lower the speed slightly The shaker should be set at the highest speed allowable without splashing of the liquid Equipment Settings FLEX
2. Support Specialist whether or not the kit is Because blood is a complex matrix which contains large numbers of proteins that may interfere with the accurate measurement of desired analytes using an optimized serum matrix in the standard curves when measuring analytes secreted in serum plasma e Significantly improves accuracy of measurement More accurately simulates the conditions of the native analyte present in serum or plasma compared to a standard curve generated by spiking an analyte into a buffer solution e Mimics the environment of native analytes in serum or plasma performing correctly Other commercial multiplex kits add a serum Individual labs can qualify their own assay matrix to sample wells With some exceptions we performance by including a high and low QC that may better reflect their unique experimental samples do not do this for the following reasons While this method does effectively show good recoveries in most cases adding serum matrix to sample wells can mask the matrix effect likely QCsare important for translational studies that affecting the sensitivity of the actual analyte ds measurement require more validation ensuring that the data are reproducible across various kit lots t is very difficult to predict the effect of mixing serum matrix with samples from a randomly sampled population QCsare also important when comparing data for multi site
3. The orbital titer plate shaker should be set at Cat No 40 097 a speed to provide maximum orbital mixing Brochure Lit No DS0012ENEU for more information without the splashing of liquid outside the wells For the recommended plate shaker this would be a setting of 5 7 which is approximately 500 800 rpm Handheld Magnetic Separator Block for 96 well Flat Bottom or Conical Well Plates Cat No 40 285 Note that orbital shakers vary Your shaker can be calibrated by pre wetting the plate with buffer and slowly increasing the speed until splashing occurs Then lower the speed slightly The shaker should be set at the highest speed allowable without splashing the liquid e uminex 200 MAGPIX or FLEXMAP 3D analyzer instruments available from Merck Millipore and manufactured by Luminex9 Corporation Sheath fluid Luminex 200 or FLEXMAP 3D systems or drive fluid MAGPIX instrument Sheath fluid and drive fluid are specific for use in the appropriate instrument and cannot be used interchangeably Sheath fluid or drive fluid can be reordered directly from Merck Millipore BioTek plate washer model 405 LS top and Sheath Fluid Cat No SHEATHFLUID MAGPIX Drive Fluid 4PK Cat MPXDF 4PK Sample Collection and Preparation General Information Proper and consistent pipetting technique is key to con
4. The Power of Biomarker Analysis Multiplex assay tips from the makers of MILLIPLEX map Merck Millipore is a division of MERCK Why just multiplex when can MILLIPLEX For over ten years Merck Millipore has offered the benefits of MILLIPLEX map multiplexed assay panels containing all the components and reagents you need to detect multiple analytes simultaneously The benefits of multiplex protein detection assays are endless but navigating a protocol can be challenging We re so confident in the benefits of MILLIPLEX map kits that we ve compiled this book of tips and tricks straight from the experts to eliminate any doubt in your ability to multiplex like a pro Every year thousands of your colleagues experience the benefits of MILLIPLEX map kits publishing in scientific journals around the world We hope this guide enhances the power of your research with multiplexing MILIPLEX py Map NOTE Alternate methods presented in this guide may deviate from the protocol These methods have either been tried by Merck Millipore or our end users using our MILLIPLEX kits We cannot guarantee methods presented will work in all cases These procedures have not been validated Table of Contents Introduction The Luminex xMAP Technology MILLIPLEX map Bring your biomarkers to life Deciding Which MILLIPLEX Assay is Best for Your Research General Assay Information Material
5. and MAGPIX instruments are built by the Luminex Corporation MILLIPLEX map kits can be run on any of these machines regardless of the name given to the machine by a Luminex business partner If using Luminex instruments with software other than xPONENT software Bio Plex Manager MasterPlex STarStation LiquiChip LABScan 100 follow instrument instructions for gate settings and additional specifications from the software vendors for reading assays using Luminex magnetic beads To read a MILLIPLEX9 kit on a Bio Plex machine select 5K 25K for magnetic beads depending on the version of Bio Plex Manager software Overview of Instrument Considerations During a MILLIPLEX9 map Assay Starting up and shutting down your system correctly will ensure its longevity The instructions for the MAPGIX and Luminex 200 systems are located within the systems user manual Short term cleaning will prevent sample induced clogging while long term cleaning is important to ensure that drive fluid does not evaporate and crystallize Preparation Check probe and insert into reader set probe height Fill reservoirs Milli O water 70 EtOH 0 1M NaOH Revive instrument revive from storage daily start up Calibrate and verify instrument system installation Read the entire kit protocol Acquire Materials Required But Not Supplied Confirm accuracy of pipettes Assay
6. 4 1 3 1 4 Panel Name dPAPP A Tissue Factor Human CVD4 1 Panel Name MMP 9 sP Selectin Mouse CVD1 4 Angiogenesis kits 6 samples run Panel Name EGF G CSF ET 1 FGF 1 Follistatin HB EGF VEGF A Human 5 Angiogenesis Growth Factor 3 5 2 Guinea Pig Four serum or plasma samples were usually run in each kit Exceptions are noted Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits Panel Name EGF MDC Human Cytokine Chemokine Panel 1 Panel Name SDF 10 B IL 16 Human Cytokine Chemokine Panel 2 4 Panel Name GM CSF Human TH17 Panel Name IL 2 Human CD8 Panel Name Eotaxin IFNy IL 1p IL 12 p40 IL 13 IL 15 IL 17A IP 10 Mouse Cytokine Chemokine Panel 1 2 2 4 4 1 1 4 Panel MIP 2 LIX RANTES Human CD8 2 Cytokine kits continued Panel Name IL 17E GM CSF IFNy IL 2 IL 4 IL 5 IL 22 IL 28B Mouse TH17 4 3 4 2 1 4 4 4 IL 10 IL 23 IL 12 p70 IL 15 IL 17A IL 17F IL 33 TNFB 1 4 4 2 4 1 4 2 sCD40L 3 Panel Name GM CSF G CSF IL 2 VEGF A Non Human Primate Cytokine 1 4 4 2 Chemokine Panel Name Eotaxin GM CSF IL 10 Leptin IL 4 IL 18 IL 2 IL 6 Rat Cytokine C
7. Follow the kit protocol Setup experimental design on acquisition software Run assay Run Post Batch Routine Shutdown Daily shutdown overnight Run Clean Routine Run Daily Shutdown Routine Remove probe and clean in a sonicating water bath Long term shutdown longer than one week Run Clean Routine multiple times Run Prepare for Storage part 1 e Prime multiple times with Milli Q9 water use an empty sheath fluid container Run Prepare for Storage part 2 Remove probe and clean in a sonicating water bath Luminex 200 User Manual Section 3 page 17 MAGPIX User Quick Guide 4 2 MILLIPLEX Analyst 5 1 Software Merck Millipore offers the most powerful combination software package including best in class multiplex data analysis MILLIPLEX9 Analyst 5 1 software coupled with data acquisition using the Luminex xPONENT software MILLIPLEX Analyst 5 1 software enables you to manage track and analyze your multiplex assays rapidly and efficiently giving you more time to focus on advancing your research Data acquisition and analysis integrates seamlessly with all Luminex instruments including FLEXMAP 3D Luminex 200 and MAGPIX systems MILLIPLEX9 Analyst 5 1 software is available in one and five seat licenses enabling complete flexibility for small medium and large laboratories Step 1 Choose the folder containing the exported data files 2013 03 07 170302
8. MFI Median Fluorescence Intensity value of the bead population However MFI will not change for bead counts greater than or equal to 35 Therefore don t worry if there is a 35 bead count on one bead region and 400 for others MFIs will not be affected How to Correct or Prevent Low Bead Counts e Be sure to specify MagPlex in the kit protocol for software or use the correct gate setting on Bio Plex software Sample preparation Thaw vortex and centrifuge samples at a minimum of 3 000 x g Avoid or remove any fat layers that may develop For samples known to be challenging e g synovial fluid saliva one may increase wash steps after incubation with primary antibody Resuspend beads in wash buffer instead of sheath drive fluid However the plate must be read within four hours Add 1x wash buffer which contains Tween 20 to keep the beads from clumping or sticking Store only in sheath or drive fluid When using a handheld magnet blot the plate gently When using a plate washer check the settings to make sure the plate is soaking for 60 seconds and the aspiration is not all the way down in the well Warm the plate to room temperature after an overnight 4 C capture antibody incubation step Let the plate shake at room temperature for one hour For MAGPIX users cleaning the instrument is critical Special care should be taken to use the enhan
9. None Date modified S gt Bato detect cay 2012 1003 103914 Fie 212KB p standard curves STD defined in onginaticev file to set up 1 C Usem 211375 Desktop WMA 5 1 tearing detect cav Step 4 Importing Data from xPONENT software using Auto Detect Curve Plate Map Assign analytes dilutions and sample names as needed e Ifthe kit and Plate Layout will be used again save as a Protocol e When ready for Analysis click Next 4 Ls s e uc 2000 am 2 Vo Eag tt h Cuem Orton 8B F 59 Eg i5 sot Lu I 1 1 T Uisant 1 Dineen Dinos Step 5 Data Analysis with MILLIPLEX Analyst 5 1 software Concentration of each analyte is calculated in pg mL using the Standard Curve Best Fitting is a 5 P log curve fitting algorithm Click on different analytes to see where that sample is on the Standard Curve All graphs are updated in real time Detailed Reports can be saved as an Excel file 11 Data Analysis Bead Counts Merck Millipore recommends counting 50 beads According to Luminex a minimum of 35 beads per region need to be counted Fewer than 35 beads could cause a shift in the
10. o 5 E Deciding Which MILLIPLEX Assay 15 Best for Your Research Merck Millipore s ELISAs and MILLIPLEX assays for the same analyte commonly use the same antibody pairs and conditions n Method Comparison tests while the absolute values are not exactly the same the results do correlate Hence when switching from one assay platform to another a correlation factor may be used when comparing with past data In most situations the dynamic range and the sensitivity of the assay may be better with the MILLIPLEX kits Please contact Technical Support for more information on correlation factors To locate protocols and technical documents for a specific panel Search the website for the catalog number The link to the protocol can be found using he Documents tab The right hand side of the product name and catalog number The top of the Product Description page The easiest way to find a panel that contains the analytes you want to measure would be to search online The MILLIPLEX Analyte Kit Finder located on the MILLIPLEX home page Search the latest edition of the Analyte Quarterly www merckmillipore com milliplex To find publications citing a specific panel or analyte Search the website for the catalog number Links to some of the references using the product can be found at the top of the Product Description page Fora more complete list contact Tech
11. the fat cake was discarded and the ho mogenate was centrifuged again at 14 000 x g for 20 minutes at 4 C The supernatant was stored in aliquots at 70 C The vessel was dissected and all the surrounding tissues removed The vessel was mixed and homogenized in a rotor stator with 1 mL of lysis buffer 0 1 g of bovine serum albumin 5 uL of Triton9 100 100 mg of gentamycin sulfate 100 uL of HEPES buffer 1M 23 pL of aprotinin 18 391 mg of sodium orthovanadate and PBS to complete 1 ml After this 2 mL of the lysis buffer was added to the content and was homogenized in a Potter Elvehjem tissue grinder This was centrifuged at 400 x g for 10 minutes at 4 C The supernatant was analyzed Plasma and brain tissue from injured hyperintense tissue on DW MRI during occlusion and anatomically matching tissue from the contralateral hemisphere were collected from control and minocycline or PBS treated rat pups following 24 hours of reperfusion The flash frozen brain tissue was homogenized in a buffer containing 20 mmol L Tris HCl pH 7 5 150 mmol L NaCl 1 mmol L PMSF 0 05 Tween 20 and a cocktail of protease inhibitors Roche and protein concentration was measured in each sample For lavage samples use 50 uL sample 25 beads in sample wells Set up standards using one additional lower point and dropping the highest concentration standard point Use a buffer matrix or medium used to collect the lavage sample as the matrix ie 25 p
12. 10 msec Interrogate label with green laser 525 nm dwell time v Identify bead Quantify region based on binding events internal dye concentrations LED based analysis Monolayer beads Magnetic capture 0 5 sec dwell time Interrogate bead with red LED 635 nm 4 Interrogate label with green LED 525 nm Identify and quantify with CCD imager MILLIPLEX map Bring your biomarkers to life MILLIPLEX mar kits offer multiplex detection of biomarkers in key research focus areas Bone Metabolism Cancer Biomarkers Cardiovascular Disease M e Immunology Immune Response Intracellular M Cell Signaling Cellular Metabolism Metabolism Endocrinology Neuroscience M Toxicity M Our kits offer M The broadest selection of analytes across a wide range of disease states for both circulating and intracellular biomarkers M e All the components and reagents you need to x detect multiple analytes simultaneously M Quality controls provided to qualify assay M e Analytically validated panels yielding consistent analyte profiles within panels Comparison of standard and QC lots to a reference lot to ensure lot to lot consistency e Panels that meet stringent manufacturing criteria to ensure batch to batch reproducibility L M M EX a E N et E o
13. 2 1 1 1 Panel Name IL 17F GM CSF IL 10 11 15 IL 17A IL 22 IL 9 Human TH17 1 4 3 1 3 1 3 9 IL 1B IL 33 IL 2 IL 4 IL 23 IL 17E 11 27 IL 31 3 1 3 3 1 2 1 TNFB 3 Panel Name GM CSF sCD137 IL 10 IL 13 Granzyme B IL 2 IL 4 10 Human CD8 1 2 1 1 1 4 1 1 1 1 Panel Name GM CSF IFNy IL 10 IL 13 IL 17A IL 1B IL 2 IL 4 Human High Sensitivity T Cell 2 1 3 1 2 1 1 3 IL 23 IL 7 IL 8 MIP10 MIP1B 2 1 3 2 1 Panel Name Eotaxin G CSF GM CSF IFNy 1 10 M CSF IL 1B IL 2 Mouse Cytokine Chemokine Panel 1 4 1 4 3 2 4 4 2 IL 3 IL 4 IL 5 IL 7 IL 10 IL 12 p40 IL 13 IL 15 2 1 3 3 1 1 3 2 IL 17A IP 10 MIP 2 KC LIF LIX MCP 1 10 2 4 2 4 2 4 4 4 1 MIG RANTES TNFa IL 12 p70 VEGF A IL 9 1 9 3 4 2 4 3 29 Appendices Cytokine kits continued Panel Name GM CSF IFNB MIP 3o IL 1B IL 2 IL 4 IL 5 IL 6 Mouse TH17 4 3 1 2 3 1 1 1 IL 21 IL 22 IL 28B IL 10 IL 23 IL 12 p70 IL 27 IL 13 4 4 3 2 1 1 0 2 IL 15 IL 17F IL 33 IL 31 TNFB TNFa sCD40L 4 4 4 1 4 3 4 Panel Name GM CSF TGFa G CSF IFNy IL 2 IL 10 IL 15 sCD40L Non Human Primate Cytokine 1 4 3 2 4 0 2 Chemokine IL 17A 1 13 IL 1B 1 4 IL 5 IL 6 IL 8 4 3 0 2 2 4 2 1 10 TNFa IL 12 VEGF A IL 18 1 3 1 4 2 Panel Name G CSF Eotaxin GM CSF IL 10 Leptin 10 IL 18 IL 2 Rat Cytokine Chemokine 1 1 3 2 4 4 4 4 IL
14. Examples of poor technique include Not vortexing between tubes Not vortexing while loading the plate Not pipetting equal amounts into the plate The lower the concentrations of analytes the higher the CVs tend to be With new users this improves with time and practice For any standard points that have high CVs samples in that range of the curve should be interpreted with caution Alternatively a standard point or one of the replicate wells can be flagged masked although it can be difficult to decide which well to flag if only duplicates are run Recovery Percent recovery should be 100 30 industry minimum although some customers will have their own acceptance criteria Percent recovery is usually worse at either extreme of the curve but this also improves with time and practice For curve statistics focus on the R value which approach but never equal unity Note that a R value of 1 is seen with software rounding of 0 9999 Minimum Maximum Detectable Concentration minDC maxDC For many assays the minDC maxDC will be outside the standard points extrapolated due to good curve performance and fit To avoid seeing extrapolated data set the desired range of detection in MILLIPLEX9 Analyst 5 1 software Deciding whether to use the Best Fit vs 5 parameter lot option depends on your comfort level to determine how appropriate it is to play with curve fit to find t
15. Folder 2013 07 10 10 25 37 Folder 2013 07 02 155804 Folder 2013 02 27 133340 Folder 2013 04 05 16 43 18 Folder 2013 06 28 092352 Folder 2013 04 09 13 48 40 Folder 2013 07 19 08 29 53 Folder 2013 06 23 08 10 07 Folder Pure 2013 07 08 122049 Folder Ma 5 1 app note 2013 06 23 145855 Folder MA 5 1 Bugs 7 1 2013 2013 07 01 151703 Folder MA 5 1 database and genomics 2013 01 14 122025 Folder J MA 5 1 database big let 2013 03 08 112052 Folder 2013 07 16 144548 Folder 2013 07 23 08 15 10 Folder 2012 12 20 112428 Folder 2012 11 05 151044 Folder 2012 05 21 15540 Folder 2013 06 27 164201 Folder m Y a ums 2012 07 19 1624100 Folder 0013 06 28 20 Step 2 Select the appropriate File Type from the pull down menu CSV Files csv from xPONENT software Excel files xIs from Bio Plex software Oste moded Sze 38 Be Rad Spot Repon Tabie te Excel 96 Welais 2012 1003 142318 Fle KB 21 22 Step 3 Importing data from xPONENT software using Auto Detect Curve Set Auto Detect Curve to STD and highlight the file auto detect csv Press the green arrow icon Loaded files are shown in the File Names window The Standard Curve settings will be ignored and the software will use the Standard Curves as set up in xPONENT software Click Next to continue to the Plate Map screen 4211375 MA 5 1 trig El ag
16. IL 12 p70 VEGF A IL 9 1 4 3 Panel Name IL 17E GM CSF IFNy IL 2 IL 4 IL 5 IL 6 IL 21 Mouse TH17 Panel 2 3 1 2 2 2 2 1 IL 22 IL 28B IL 10 IL 23 IL 27 IL 13 IL 15 IL 17A 4 4 4 2 1 3 2 1 IL 33 TNFB sCD40L 2 4 1 Panel Name Leptin IL 18 IL 2 IL 13 IL 10 IL 4 MCP 1 IP 10 Rat Cytokine Chemokine 4 4 1 1 3 2 1 4 GROo KC VEGF A Fractalkine LIX MIP 2 RANTES 4 B 2 1 3 2 Panel Name GM CSF IL 1B IL 1ra sCD40L IL 17A IL 12 IL 18 Porcine Cytokine Chemokine 3 3 1 3 2 4 1 Panel Name TGFa G CSF IFNy IL 2 IP 10 IL 13 IL 5 IL 6 Non Human Primate Cytokine 1 2 3 4 4 2 4 2 Chemokine 1 12 23 IL 8 10 MCP 1 TNFa p40 VEGF A 1 1 3 1 1 3 Panel Name IL 18 Canine Cytokine Chemokine 2 CVD kits Panel Name FABP3 Troponin Human CVD1 4 4 Panel Name ADAMTS13 D Dimer FABP5 GDF 15 MPO sP Selectin Human CVD2 4 4 4 4 4 1 Panel Name dPAPP A Tissue Factor Troponin T Human CVD4 3 1 3 Panel Name MMP 9 PAI 1 total sP Selectin Mouse CVD1 1 1 4 Angiogenesis kits 6 samples run Panel Name BMP 9 FGF 1 Follistatin Human Angiogenesis Growth Factor 3 3 2 e Four serum or plasma samples were usually run in each kit Exceptions are noted e Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits Panel Name EGF Eotaxin Fractalkine G CSF GM CSF GRO 2 IFNy Human Cytokine Ch
17. Information Deliver extremely precise volumes of solvent when reconstituting lyophilized products Variations of even a few microliters will significantly affect quantitation Do not mix or substitute assay reagents with those from other lots or sources e f leftover reagent lots match and the reagents have been kept at the appropriate storage conditions they can be used in combination until the expiration dates Serum matrix bead diluents and wash and assay buffers from other kits can be used combined if the catalog numbers of these components match in the protocols for the kits in question Use of Bead Diluent Approximately 10 of a normal population of samples especially human serum or plasma samples has heterophillic antibodies that can nonspecifically bind to the capture and detection antibodies simultaneously thus generating a false positive signal Bead diluents contain a cocktail of proprietary reagents that significantly reduce this false signal without reducing the true analyte measurement Bead diluents may also contain factors for detection e g insulin in mouse kits f assay buffer was erroneously added instead of bead diluent transfer samples to a clean clear centrifuge tube spin down and remove buffer replace with bead diluents and proceed with the assay protocol NEC Se rgo Antibody Immobilized Beads The antibody immobilized beads are light sensitive and must be
18. buffer for your assay and if sample has not yet been loaded remove wash buffer and replace Replace the detection antibody cocktail volume with assay buffer with assay buffer add SAPE and continue to follow the protocol f no detection antibody is available add SAPE and continue to follow the protocol keeping in f sample has been added to the plate with wash buffer there is a potential for low recovery as it may not have the required protein concentration or protease inhibitors mind that the signal may be lower Vortex all reagents well before adding them to The plate should be read immediately within the plate 4 hours after the assay is finished If the plate cannot be read immediately seal the plate cover with aluminum foil or an opaque lid and store the When using frozen samples it is recommended to plate at 2 8 for up to 72 hours with samples thaw the samples completely mix well by vortexing ro ghtup There fay Deis at high setting and centrifuge at a minimum is loss of sensitivity after 24 hours of 3000 x g prior to use in the assay to remove particulates Before reading the plate agitate the plate on the plate shaker at room temperature for 10 minutes Be precise when adding samples standards and QCs to the plate Pipette to the sides of the wells Be sure all fluid is expressed out of the pipette tips e Itis possible to run a portion of
19. countries across Europe please call 44 0 115 943 0840 Or visit www merckmillipore com offices For Technical Service visit www merckmillipore com techservice Get connected Join Merck Millipore on your favorite social media outlet for the latest updates news products innovations and contests f facebook com MerckMillipore twitter com MerckMillipore Distributed by Abacus ALS Free Call 1800 222 287 AUS 0800 222 170 NZ Email info abacus als com Web www abacus als com
20. i e Radioimmunoprecipitation assay RIPA buffer then cell lysate must be diluted to less than 0 05 SDS for assays to detect intracellular proteins such as cell signaling proteins Note to solubilize nuclear mitochondrial proteins you must use either SDS or another method such as ultrasonication to puncture the tough nuclear mitochondrial membranes e Reducing agents like B mercaptoethanol or dithiothreitol are not recommended Maximum allowed protein Luminex assay Type of detergents Protein localization concentration compatibility Non ionic detergents Cytoplasm 5 mg mL Yes detergents Membrane bound SL lonic detergents Membrane bound Nucleus 5 mg mL Requires dilution Mitochondria 28 Perform all dilutions with lysis buffer not assay buffer or phosphate buffered saline PBS Total protein concentration limits Do not collect lysates at greater than 5 mg mL protein concentration At protein concentrations higher than 5 mg mL not all proteins will be solubilized equally by the lysis buffer Some proteins can be solubilized at a given detergent concentration while other proteins are not as affected For example B tubulin signal decreases with increasing total protein concentration signal decrease occurs at 5 to 6 mg mL for Jurkat cell and peripheral blood mononuclear cell PBMC lysates Total protein concentrations should be within a specific range which is outline
21. protected from light at all times Cover the assay plate containing beads with an opaque plate lid or aluminum foil during all incubation steps Any unused mixed antibody immobilized beads may be stored in the Bead Mix Bottle at 2 8 C for up to one month Standards After hydration reconstitution all standards and controls must be transferred to polypropylene tubes During the preparation of standard curves thoroughly mix each higher concentration before making the next dilution The standards prepared by serial dilution must be used within one hour of preparation Discard any unused standards except the standard stock The standard stock can be stored at lt 20 C for one month or at lt 80 for more than one month Standard 100 uL 100 100 uL 100 uL 100 uL 100 uL w Ww sand 3 M 2 S k AT 49 0 qal 469 088 gal The quality of the standard curves can be determined by the recovery of the standards and the QC values Quality Controls e We include Quality Controls OCs to qualify assay performance QC values are based on a minimum of six assays run by at least three different operators The midpoint and 35 of the high low value mean are reported When customer contacts Technical Support with a concern related to assay performance the customer is usually first asked if the values are in a specific range This tells the Technical
22. studies or to compare assay results from multiple technicians Kits designed for non serum plasma samples 0 urine CSF or samples that require a significant dilution at least 1 20 do not require serum matrix Effect of Serum Matrix f the recovery of analytes spiked into sample wells e For non serum plasma samples the appropriate in an assay using a buffer standard curve falls outside our acceptance criteria 80 120 this indicates that there isa nonspecific matrix effect from the samples To compensate for this effect a serum matrix with a similar effect is added to the standard curve wells to shift the actual curve so that it matches the recovery in the sample wells Serum matrix is usually a similar sample with all the endogenous and cross reacting analytes extracted medium e g cell culture medium should be added instead of serum matrix In the absence of appropriate medium or when using a blank assay buffer can be used For cell tissue homogenates the final cell or tissue homogenate should be prepared in a buffer that has a neutral pH contains minimal detergents or strongly denaturing agents and has an ionic strength close to physiological concentrations An approximately 1 20 dilution would be required to eliminate the need for using serum matrix in an assay Enough Assay Buffer Wash Buffer e Additional assay buffer that is required for samples Incomplete washing
23. the reporter molecule to complete the reaction on the surface of each microsphere Merck Millipore provides three Luminex instruments to acquire and analyze data using two detection methods see Figure 1 e The Luminex 200 and FLEXMAP 309 systems flow cytometry based instruments that integrate key xMAP detection components such as lasers optics advanced fluidics and high speed digital signal processors The MAGPIX analyzer is a CCD based instrument that integrates key xMAP capture and detection components with the speed and efficiency of magnetic bead processing Each individual microsphere is identified by its bead signature and the result of its bioassay is quantified based on fluorescent reporter signa Merck Millipore combines the streamlined data acquisition power of Luminex xPONENT acquisition software with sophisticated analysis capabilities of the new MILLIPLEX Analyst 5 1 software integrating data acquisition and analysis seamlessly with all Luminex instruments S The capability of adding multiple conjugated beads to each sample results in the ability to obtain multiple assay results from each sample Open architecture xMAP technology enables the multiplexing of many types of bioassays reducing time labor and costs over traditional methods Flow cytometry based analysis Sheath fluid hydrodynamic focusing of sample Interrogate bead with red laser 635 nm v v
24. then comparing with your chart later beads MicroPlex Be sure to select the correct setting in the protocol for your bead type e f the wrong type is selected the plate does not need to be reread The batch can be replayed with the corrected protocol setting e The link for xPONENT software templates e www merckmillipore com Imx xponent Life Science Research gt Protein Detection and Quantification Luminex Multiplexing Instruments Multiplex Assay Analysis Software gt xPONENT If a plate cannot be run immediately within 4 hours e 9 it needs to be taken to another site to run the assay suspend your sample in sheath or drive fluid or assay buffer 10 12 plates can be run with one bottle of drive fluid for the MAGPIX system To change a standard curve from for example a 7 point curve to an 8 point curve simply make a new protocol and replay the batch Running MILLIPLEX map Kits on Other Luminex Instruments A Luminex 100 system with IS software Luminex 200 FLEXMAP 3D or MAGPIX instrument is required to run a MILLIPLEX map assay f you want to try a kit before purchasing an instrument ask your Sales Specialist to provide a demonstration using the Luminex technology and MILLIPLEX mar kits e Magnetic bead assays cannot be run on any instruments using Luminex IS 2 3 or Luminex 1 7 software Since all Luminex machines Luminex 200 FLEXMAP 3D
25. 0 cells well and onic detergents 5 mg mL Requires dilution allow growth for 48 hours e For suspension cell lines seed 250 000 cells well e Total protein concentration limits and collect at desired time gt lt 29 m D E 5 E E Do not collect lysates at greater than 5 mg mL protein e For cell lysis add 30 uL lysis buffer per well and concentration pipet up and down thoroughly without creating At protein concentrations higher than 5 mg mL not all too many bubbles For a more detailed protocol proteins will be solubilized equally by the lysis buffer request information from Technical Support Some proteins can be solubilized at a given detergent concentration while other proteins are not as affected Add protease inhibitors and or phosphatase For example B tubulin signal decreases with increasing inhibitors to home brew lysis buffers total protein concentration signal decrease occurs at 5 to 6 mg mL for Jurkat cell and peripheral blood mononuclear cell PBMC lysates Lysis buffer selection Total protein concentrations should be within a specific range Lysis buffer can be found in the Cell Signaling Buffer amp Detection Kit Cat No 48 602MAG or sold separately Cat No 43 040 Non ionic detergents 40 Tergitol IPEGAL are recommended in lysis buffers for solubilizing which is outlined in each protocol Astarting protein amount is 10 u
26. 15 1 55 63 PMID 9792497 While these alternate methods have been tried by Merck Millipore or our end users using MILLIPLEX map kits we cannot guarantee methods will work with all samples These procedures have not been analytically validated Sample Type Species Kit Run Procedure Reference if available Cervical Saliva cervical and vaginal secretions were collected using ophthalmalic sponges J Clin Immunol 1997 Secretions Wek Cel Xomed Treace Orlando FL after exposure of the cervical os with the specu Sep 17 5 370 9 Vaginal lum The secretions were collected by placing the ophthalmalic sponge directly into the PMID 9327336 Secretions and Saliva Secretions Colorectal Tissue Extracts Ear Lysates Ear Lysates Infectious Samples Jejunal Extracts Lipemic Samples Lymph Node Homogenates Saliva Human Human Cytokine Panel 1 Mouse Mouse Cytokine Panel 1 Mouse Mouse Cytokine Panel 1 Human Human Cytokines Mouse Mouse Cytokine Panel 2 Human cervical os and allowing it to absorb secretions for approximately 1 minute Vaginal secretions were collected by placing the ophthalmalic sponge against the vaginal wall and allowing the sponge to collect secretions In a similar fashion saliva was collected by placing the ophthalmalic sponge over the parotid duct and allowing the sponge to absorb saliva All sponges were immediately placed on ice and then frozen at 20 C The secretions wer
27. 1B 1 4 IL 5 1 6 IL 8 4 4 0 4 3 2 3 4 MCP 1 TNFa MIP 18 IL 12 VEGF A IL 18 1 1 2 1 2 3 GM CSF IL 1o Leptin 10 IL 4 IL 1B IL 2 Rat Cytokine Chemokine 2 2 2 4 4 3 3 3 IL 6 EGF IL 13 IL 10 IL 12 p70 IL 18 MCP 1 IP 10 1 2 2 3 3 3 2 4 GROo KC VEGF A Fractalkine LIX MIP 2 TNFa RANTES 3 4 4 3 4 2 2 CVD kits 8 samples run Panel Name NT proBNP CK MB CXCL6 Endocan 1 Oncostatin Troponin I Human CVD1 1 8 2 2 2 5 Panel Name ADAMTS13 D Dimer FABP5 GDF 15 Myoglobin sP Selectin Lipocalin 2 Human CVD2 8 3 3 8 7 8 1 SAA 4 2 Panel Name Macroglobulin CRP Fetuin A AGP Fibrinogen sL Selectin Haptoglobin Human CVD3 4 1 1 4 2 4 4 Platelet von Willebrand Factor 4 Factor 1 4 Panel Name Tissue Factor Troponin T Human CVD4 5 7 Panel Name sP Selectin Mouse CVD1 1 Angiogenesis kits 6 samples run Panel Name EGF ANGPT 2 ET 1 FGF 1 IL 8 HB EGF PLGF Human 1 5 5 5 1 1 VEGF C VEGF D FGF 2 VEGF A 1 4 5 1 Rabbit Four serum or plasma samples were usually run in each kit Exceptions are noted Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits Panel Name EGF Eotaxin FGF 2 G CSF GRO IFNy IL 10 IL 1B Human Cytokine Chemokine Panel 1 4 4 4 2 4 4 3 4 IL 1ra IL 3 IL 4 IL 5 IL 6 IL 7 IL 8 IL 9 2 1 1 7 2 2 2 3 IL 10 IL 12 p40 IL 12 p
28. 6 EGF IL 13 IL 10 IL 12 p70 IFNy IL 17A IL 18 2 4 3 4 2 2 1 4 1 IP 10 GROo KC VEGF A Fractalkine LIX MIP 2 TNFa 3 4 3 4 4 4 3 2 RANTES 1 Panel Name IL 1 IL 1 IL 1ra IL 2 IL 4 IL 6 IL 10 IL 12 Porcine Cytokine Chemokine 1 4 4 4 4 2 4 4 IL 18 4 Cardiovascular Disease CVD kits Panel Name NT proBNP CK MB CXCL6 Endocan 1 FABP4 LIGHT Oncostatin Human CVD1 4 4 1 1 1 1 Panel Name ADAMTS13 D Dimer FABP5 GDF 15 Myoglobin sP Selectin sVCAM 1 SAA Human CVD2 4 3 4 4 3 1 2 Platelet von Willebrand Panel Name Macroglobulin AGP Fibrinogen sL Selectin Haptoglobin Factor 4 Factor Human CVD3 4 3 1 4 4 2 4 Panel Name Follistatin dPAPP A sPECAM 1 Pentraxin 3 Tissue Factor Thrombomodulin Troponin T Human CVD4 3 4 2 4 1 1 8 Panel Name MMP 9 Mouse CVD1 1 Angiogenesis kits 8 samples run Panel Name EGF ANGPT 2 Leptin FGF 1 IL 8 HGF HB EGF VEGF C VEGF D FGF 2 VEGF A Human 3 8 1 8 1 2 8 2 6 2 2 Angiogenesis Growth Factor 30 Feline Four serum or plasma samples were usually run in each kit Exceptions are noted Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits Panel Name EGF Eotaxin FGF 2 Fit 3L Fractalkine G CSF GRO 2 Human Cytokine Chemokine Panel 1 1 1 1 1 2 2 2 2 IFNy IL 10 IL 18 IL 1ra IL 2 IL 3
29. 7 OOR Unknown12 3 7 1 0e 07 OOR Unknown13 3401 1 0e 07 OOR Unknown14 3401 1 0e 07 OOR Unknown15 5 0 1 0e 07 OOR Unknown16 16 8 8 0 OOR Unknown17 24 0 22 0 OOR Unknown18 28 6 29 0 1 1 Unknown19 3401 1 0e 07 OOR lt Unknown20 3401 1 0e 07 OOR Unknown21 3401 1 0e 07 OOR lt Unknown22 14 7 3 0 OOR lt Unknown23 3401 1 0e 07 OOR lt Unknown24 3401 1 0e 07 OOR lt Unknown25 6 8 1 0e 07 OOR lt Unknown26 3401 1 0e 07 OOR lt Breen Exirapolsted value Unknown27 3401 1 0e 07 OOR Orange Extrapolated value Unknown28 5 9 1 0e 07 OOR OOR lt Out of Range Below Unknown29 4 5 lt 1 0e 07 OOR lt lt 3 40 1 Out of Range Below Unknown30 8 2 lt 1 0e 07 OOR lt 1 0e 07 Out of Range Below Unknown31 3401 1 0e 07 OOR Best Fitting 5P Log Unknown32 3401 1 0e 07 OOR Table 2 Significantly more IFN y concentrations could be calculated at the low end of the curve in the Rat Cytokine Chemokine Magnetic Bead Panel by MILLIPLEX9 Analyst 5 1 software compared to the Bio Plex and StatLIA software packages Intracellular Assays 12 Differences between multiplex assays for circulating analytes vs intracellular analytes Circulating Analyte Assay Intracellular Analyte Assay Quantitative Qualitative fold change Serum plasma tissue culture urine CSF etc Cells must be lysed Analytes analytically validated within panel Fixed kits and individu
30. 70 IL 13 IL 15 IL 17A IP 10 MCP 1 3 2 0 2 2 3 1 1 MCP 3 MDC 10 1 sCD40L TGFa TNFB VEGF A 2 4 4 2 4 4 4 3 PDGF AA 4 Panel Name MCP 2 MCP 4 ENA 78 SDF 1o p 1 309 TARC 6Ckine Eotaxin 3 Human Cytokine Chemokine Panel 2 0 0 0 3 2 1 3 3 CTACK IL 23 LIF TSLP IL 33 1 3 3 3 3 Panel Name GM CSF IL 17A Human TH17 4 4 Panel Name GM CSF sCD137 IFNy IL 10 IL 6 Human CD8 2 4 4 2 4 Panel Name IFNy IL 10 M CSF IL 18 IL 2 IL 3 IL 4 IL 5 Mouse Cytokine Chemokine Panel 1 1 2 2 1 1 1 1 1 IL 12 p40 IL 15 IL 17A IP 10 MIP 2 LIF LIX 10 4 1 1 2 B 1 3 1 1 RANTES TNFa VEGF A IL 9 2 4 1 4 2 39 40 Cytokine kits continued Panel Name IL 17E GM CSF IFNy IL 2 IL 4 IL 5 IL 6 Mouse TH17 2 4 9 1 3 1 1 4 IL 22 IL 28B IL 10 IL 23 IL 12 p70 IL 27 IL 13 IL 15 4 4 3 2 2 9 1 1 IL 17A IL 17F IL 33 IL 31 TNFB sCD40L 3 1 2 2 4 Panel Name GM CSF TGFo G CSF IL 2 IL 17A IL 13 IL 5 IL 8 Non Human Primate Cytokine 0 4 2 4 4 1 1 4 Chemokine 10 VEGF A 2 4 Panel G CSF GM CSF IL 10 Leptin 10 IL 4 IL 1B IL 2 Rat Cytokine Chemokine 1 3 3 4 3 3 3 3 IL 6 EGF IL 13 IL 10 IL 12 p70 IFNy IL 17A IL 18 2 2 3 4 2 2 1 4 MCP 1 IP 10 GROo KC VEGF A Fractalkine LIX MIP 2 RANTES 2 4 4 3 3 3 4 1 Panel Name IFNy IL 18 TNFa Canine Cytokine Chemokine 4 3 1 Panel Name IL 4 IL 18 Porcine Cytokine Chemokine 4 1 Appendix 2 Sample Preparation M
31. G 60K 05 and list Phone 512 381 4397 Toll free 1 877 785 2323 Fax 512 219 5114 the specific analytes How to design and order a customized kit online e Email support luminexcorp com From the Product Description page Click Design amp Purchase Your Own Kit Make your choices add to the cart and or e For questions or issues with Biolek washers contact BioTek at save to your favorites and go to Checkout All Regions e From the MILLIPLEX map website www merckmillipore com milliplex Technical Support Click Design amp Purchase Your Own Kit In North America Call 800 242 4685 Outside the U S Call 802 655 4740 www merckmillipore com biotek contact Make your choices add to the cart and or n save to your favorites and go to Checkout n Quick Purchase Click on the Begin icon located Email TAC biotek com within Design and Purchase your PR M NT MILLIPLEX9 wap Kits For questions or issues with MILLIPLEX assays Make your choices add to the cart and or please contact Merck Millipore Technical Support save to your favorites and go to Checkout or your Sales Specialist RADI SENE O The expiration date for a kit is that of the component with the shortest expiration date This date is printed on the box label Kits will ship with a minimum of 3 months until expirati
32. G H M R e e RPS6 Total 46 715MAG H M R e e Src Tyr419 46 710MAG H M R e Src Total 46 709MAG H MR e e Recommended STATI Tyr701 46 655MAG HM e s Accepit STAT Total 46 654MAG HM e EIN STAT3 Tyr705 46 623MAG e e Human STAT3 Ser727 46 624MAG H M R e Total 46 625MAG H M R o e M Mouse 5 Tyr694 Tyr699 46 641MAG H M R e e R Rat Tie2 pan Tyr 46 7 16MAG H 1 Assay Buffer 1 Tie2 Total 46 717MAG H AB2 Assay Buffer 2 VEGFR2 pan Tyr 46 7 18MAG H VEGFR2 Total 46 719MAG H o Glossary Accuracy Data representing mean percent recovery of spiked standards ranging from low medium and high concentrations in serum matrices for a defined number of samples see Spike Recovery Analyte A chemical substance that is the subject of chemical analysis Configurable customizable kit A type of MILLIPLEX mar kit that enables the customer to choose the analytes within a specific panel that best meet her research needs e g Cat No HCYTOMAG 60K Human Cytokine Chemokine Panel 1 Drive fluid Luminex Drive Fluid is the delivery medium to transport the sample to the optic components of the MAGPIX system Fixed kit A type of MILLIPLEX9 map kit that is not configurable All of the analytes are sold together with the capture beads already premixed e g Cat No 48 611 Akt mTOR Phosphoprotein 11 Plex FLEXMAP 3D system A Luminex9 i
33. IL 5 IL 6 1 2 3 2 2 2 1 1 IL 7 IL 8 IL 9 IL 10 IL 12 p40 IL 13 IL 15 IL 17A 1 1 1 2 1 2 2 1 IP 10 MCP 1 MCP 3 MDC MIP 10 MIP 1 sCD40L TGFa 1 1 2 2 2 1 2 2 TNFa TNFB VEGF A PDGF AB BB 0 2 1 Panel Name SDF 10 8 1 309 IL 23 TPO IL 33 Human Cytokine Chemokine Panel 2 3 1 2 Panel Name GM CSF IL 2 10 Human CD8 1 1 Panel Name IL 17E GM CSF MIP 30 IL 1B IL 2 IL 4 IL 5 IL 21 Human TH17 3 3 1 1 1 2 2 3 IL 22 IL 28B IL 10 IL 23 IL 12 p70 4 4 3 2 4 Panel 5 TGFa G CSF IFNy IL 2 IL 15 IL 17A IL 1ra Non Human Primate Cytokine 1 3 3 2 S 1 2 9 1 13 1 18 1 4 IL 5 IL 6 IL 8 MIP 10 TNFa 2 3 3 3 2 3 1 2 IL 12 23 1 40 VEGF A Fractalkine LIX MIP 2 TNFa RANTES 0 1 3 4 4 4 2 2 Panel Name G CSF Eotaxin GM CSF IL 10 Leptin 10 IL 4 IL 1B Rat Cytokine Chemokine 2 3 4 3 4 4 3 4 IL 2 IL 6 EGF IL 13 IL 10 IL 12 p70 IFNy IL 5 3 2 4 2 4 3 2 2 IL 17A IL 18 MCP 1 IP 10 GROo KC 2 3 3 4 4 Panel Name GM CSF IFNy IL 2 IL 6 IL 7 IL 8 IL 15 IP 10 Canine Cytokine Chemokine 4 2 4 4 3 1 3 3 KC like IL 10 IL 18 MCP 1 TNFa 3 1 4 4 1 Panel Name GM CSF IL 10 Porcine Cytokine Chemokine 31 CVD kits FABP3 FABP4 Panel Name Troponin l Human CVD1 4 Panel Name ADAMTS13 FABP5 GDF 15 Myoglobin sP Selectin Human CVD2 4 4 1 0 2 Platelet Factor von Willebrand sL Selectin SAP Panel Name Macroglobulin AGP Haptoglobin 4 Factor Human CVD3 4
34. ILLIPLEX Kits Requiring Special Sample Preparation Kit Name Sample Type Cat No Sample Trt Inhibitors Inhibitor Source Canine Gut Hormome Magnetic Bead Panel SER PLA CCS CGTMAG 98K DPP IV See Note 1 AEBSF See Note 2 Human Gut Hormone Magnetic Bead Panel SER PLA CCS HGT 68K DPP IV See Note 1 AEBSF Protease Cocktail See Notes 2 4 Human IGF 1 IGF 2 Magnetic Bead Panel SER PLA CCS HIGFMAG 52K Extraction NONE Human IGF Binding Protein IGFBP SER PLA CCS HIGFBMAG 53K Protease Inhibitor Cocktail See Note 5 Magnetic Bead Panel Human Metabolic Hormone Magnetic SER PLA CCS HMHEMAG 34K DPP IV See Note 1 Bead Panel Aprotinin AEBSF Protease See Notes 2 3 4 Cocktail Human Neuropeptide Magnetic Bead Panel SER PLA CCS HNPMAG 35K Extraction NONE Mouse Gut Hormone Magnetic Bead Panel SER PLA CCS MGTMAG 78K DPP IV See Note 1 Mouse Metabolic Hormone Magnetic SER PLA CCS MMHMAG 44K DPP IV See Note 1 Bead Panel Aprotinin AEBSF Protease NONE Cocktai Non Human Primate Metabolic Magnetic SER PLA CCS NHPMHMAG 45K DPP IV See Note 1 Bead Panel Aprotinin AEBSF Protease See Notes 2 3 4 Cocktai Rat Metabolic Hormone Magnetic Bead Panel SER PLA CCS RMHMAG 84K DPP IV See Note 1 Aprotinin AEBSF Protease See Notes 2 3 4 Cocktai Rat Mouse Neuropeptide Magnetic Bead Panel CSF RMNPMAG 83K NONE Extraction Human Skin Magnetic Bead Panel SER PLA CCS SKINMAG 50K Validated for skin tape NO
35. L standard control blank 25 uL assay buffer medium 25 uL beads The first incubation with standard sample should be overnight 4 C Final results should be divided by 2 Two 3 2 mm 1 8 inch diameter disks were punched from dried blood spot calibrators or controls and eluted in 100 uL of 0 075 mol L sodium barbital buffer pH 8 6 con taining 0 5 g L anilinonaphthalenesulfonic acid and 0 5 g L sodium azide by sonication at room temperature for 30 minutes The volume of blood per 3 2 mm disk was 3 uL 3 The eluate was filtered in a 0 45 um centrifugal filter unit Merck Millipore Whole blood samples were spotted onto Whatman 3 mm filter paper air dried and stored at 4 C prior to extraction and testing Areas equivalent to 25 drop were punched from the filter paper and eluted in 25 uL of 0 01 M phosphate buffer pH 7 4 prior to analysis The protein content of each eluate was measured spectrophotometrical ly at 260 280 nm and the samples normalized to a standard protein content of 1 ug mL J Clin Endocrinol Metab 2001 Dec 86 12 5973 80 PMID 11739472 Diabetologia 2008 Nov 51 11 2041 8 doi 10 1007 s00125 008 1126 5 Epub 2008 Aug 19 PMID 18712345 Physiol Res 2010 59 1 79 88 Epub 2009 Feb 27 PMID 19249917 BMC Cardiovasc Disord 2009 Feb 17 9 7 doi 10 1186 1471 2261 9 7 PMID 1922285 J Cereb Blood Flow Metab 2005 Sep 25 9 1 138 49 PMID 15874975 J Chromatogr B Biomed Sci Appl 1998 Sep 11 7
36. MAP 3D System Luminex 100 200 System MAGPIX System For the MAGPIX system choose the enhanced e Use the manufacturer s gate settings startup setting instead of the common startup This will ensure proper calibration and cleaning The Luminex 200 system s xPONENT 3 1 prior to running the assay acquisition software has two functions one for e Working with serum is dirtier than other magnetic MagPlex and one for nonmagnetic samples and can affect the performance of the instrument unless it is properly cleaned Luminex can provide a recommended protocol for maintenance Be sure the needle probe is clean This may be achieved by sonication and or alcohol flushes Probe height When reading an assay on a Luminex 200 instrument adjust the probe height according to the protocols recommended by Luminex to the kit solid plate using 3 alignment discs When reading an assay on a FLEXMAP 3D System adjust probe height according to the protocols recommended by Luminex to the kit solid plate using 1 alignment disc When reading an assay on a MAGPIX system adjust the probe height according to the protocols recommended by Luminex to the kit solid plate using 2 alignment discs Annotating control wells on the instrument can be tedious with a lot of manual typing It is possible to enter the wells as unknowns instead of controls to avoid typing in the annotations for controls
37. NE Steroid Thyroid Hormone Magnetic Bead Panel SER PLA CCS SITHMAG 21K Extraction NONE TGFB1 single plex Magnetic Bead Panel SER PLA CCS TGFBMAG 64K 01 Acidification NONE TGFB1 2 3 Panel Magnetic Bead Panel SER PLA CCS TGFBMAG 64K 03 Acidification NONE Note 1 DPP IV Merck Millipore Cat No DPP4 010 is used at 10 uL per mL of blood 2 Pefabloc or AEBSF Merck Millipore Cat No A8456 is used at 1 mg mL in blood 3 Aprotinin Trasylol used at 500 KIU mL of blood Merck Millipore Cat No 7107 01 4 Protease Inhibitor Cocktail Merck Millipore Cat No P2714 5 Active and Total cannot be run together in the same assay Appendix 3 Other Sample Types Protocols Using Other Sample Types Procedure Reference if available Sample Type Species Kit Run Adipose Tissue Human Homogenates Adipose Tissue Human Homgenate Apolipoprotein Panel Adipose Tissue Human CVD Panel Extract Aorta Tissue Extract Guinea Pig Human Cytokine Chemokine Panel 1 Brain Tissue Extract Rat Rat Cytokine Bronchoalveolar Lavage BAL Samples Dried Blood Spot Samples Dried Blood Spot Samples Approximately 4 g of adipose tissue from each subject was homogenized in 16 mL of ice cold deoxygenated homogenization buffer containing 10 glycerol 150 mM NaCl 2 mM EDTA 1 mM PMSF 25 mM benzamidine 10 uM leupeptin 2 5 umol L pepstatin A and 50 U mL aprotinin in 10 mM Tris HCI pH 7 0 with fou
38. Up to eight MAPmate assays single plex and growth for 8 hours the Cell Signaling Buffer and Detection kit can be For suspension cell lines seed 250 000 cells combined into a custom multiplex kit well collectat desired time Refer to the guidelines provided in the e For cell lysis add 30 pL lysis buffer per well and MAPmate protocol pipet up and down thoroughly without creating too many bubbles For a more detailed protocol request info from Technical Support t MAPmate assays beaded Unbroken cells parts can be cleared by either intracellular assay kits to enhance the panel or filtration or by centrifugation serve as controls Refer to the guidelines provided in the kit protocol Add protease inhibitors and or phosphatase inhibitors to home brew lysis buffers Lysis buffer can be found in the Cell Signaling Buffer amp Detection Kit Cat No 48 602MAG or it is sold separately Cat No 43 040 Other lysis buffer selections Non ionic detergents 40 Tergitol IPEGAL are recommended in lysis buffers for solubilizing cytoplasmic proteins e Partially ionic detergents Triton X 100 are recommended in lysis buffers for cytoplasmic or membrane bound proteins detergents sodium dodecyl sulfate SDS are recommended in lysis buffers for membrane bound nuclear or mitochondrial proteins If using SDS in the lysis buffer
39. a plate initially IPIE PII ERICH EP then reuse the plate with other samples later For incubating assays overnight a power supply must Cover the wells that are not being used be available for the orbital shaker in a refrigerator or Use precise volumes of reagents to ensure that cold room enough remains to run the remaining wells at a gt G d gt D e Ifthe plate shaker has been turned off during the later time e Store reagents at appropriate conditions quickly after the first use e g stock standard at 20 C or lower night shake again at room temperature for one hour before proceeding with the assay protocol Remake standards for subsequent batches Be After overnight incubation of assays remember to 9 y sure to run a standard curve for each batch allow all reagents to warm to room temperature When running subsequent batches cover the 20 C 25 C before use in the assay previously used wells The mix of beads may be used for one month Detection antibody cocktail and SAPE incubation if stored at 2 8 C stock standards should be times are critical Do NOT exceed the dictated times stored at 20 C for one month and at x 80 C as this will result in higher background signals for more than one month 4
40. al MAPmates are analytically validated Kit includes standards and OCs Kits and MAPmate assays include positive and negative control cell lysates Most panels are customizable Kits are fixed create custom kits by combining Cell Signaling MAPmate assays The following MAPmate assays should not be Using Cell Signaling plexed together MAPmate Assays Phospho specific and total MAPmate pairs All magnetic MAPmate assays require the Cell Signaling Buffer amp Detection Kit Cat No 48 602MAG This kit contains all necessary reagents except the MAPmate assays Both a filter and flat bottom plate are included for convenience To select the appropriate buffer for your MAPmate assays please refer to the protocols or the buffer selection tables on the website in the Analyte Quarterly or Appendix 5 e g total GSK3B and phospho GSK3B Ser9 Pan Tyr and site specific MAPmate assays e g phospho EGF Receptor pan Tyr and phospho STAT1 Tyr701 More than 1 phospho specific MAPmate assay for a single target e g pAkt Ser473 and pAkt Thr308 GAPDH and B Tubulin assays cannot be plexed with kits or MAPmate assays containing pan Tyr assays www merckmillipore com cellsignaling_assays Preparation of Cell Lysates for Intracellular Assays Plexing Cell Signaling e 96 well plates MAPmate Assays For adherent cell lines seed 40 000 cells well
41. can adversely affect the assay requiring higher dilutions may need to be ordered outcome before running the assay See Protocol sections ee Reagents Supplied or Replacement Reagents for All washing must be performed with the wash the appropriate catalog number buffer provided f more is required see Protocol sections Reagents Supplied or Replacement Reagents for the appropriate catalog number Immunoassay Procedure 7 e Cover the plate with a plate sealer before shaking Tips for Reducing Variability Ensure proper sample collection e The plate shaker speed should be increased to PPE TALON LIN agitate the plate at the highest speed that does not To avoid low bead counts thaw vortex and lead to splashing on the sealer centrifuge all samples for 5 10 minutes at a minimum of 3 000 x g Avoid or remove any fat Immunoassay Procedure layers that may develop Before running an assay check the instrument the night before Centrifuge samples after thawing or if they appear js ihednstrument calibrated turbid This is especially recommended for plasma Has itbcen maintained samples e Have fresh water prepared calibrated and E EEE EE EA accurate pipettes multichannel pipettes andian Ensure the proper mixing of samples and controls orbital shaker or alternative VERE LORETTA RASA AS RD HT ONES e Confirm availability of a cold ro
42. ced startup or washing procedures There is an advanced cleaning method that includes sodium hydroxide NaOH and bleach Washing between wells can also be selected during the plate reading Cleaning the instrument regularly is important even if the instrument is not being used Percent Coefficient of Variation CV High CVs for standards or samples can be due to bead count For assays that have a standard curve our target inter assay CV is lt 15 and our target intra assay CV is lt 10 For qualitative assays no standard curves the MFI target inter assay CV is lt 20 and the target intra assay CV is lt 15 Calculating Lower Limit of Quantification LLOQ and Upper Limit of Quantification ULOQ Defining and ULOO requires a tight standard curve e g 1 2 or 1 3 serial dilution to the point that you achieve saturation at both ends Choose the lowest and highest standard curve points that have a recovery of 20 e Verify that this is the LLOQ and ULOQ by running 5 assays with the LLOQ and ULOQ as samples against a curve using the assay serial dilution factor where the lowest standard is below and the highest standard is above ULOQ e Inter assay precision should be within 20 for and 0100 samples Curve Performance Fit e Standard point CVs should be lt 15 High CVs here indicate improper technique was used when making standard curve dilutions
43. d in each protocol Astarting protein amount is 10 ug per well 10 ug protein in the final 25 uL that is loaded into each assay well is recommended Working backwards from there 10ug 25 pL 0 4 pg pL mg mL Diluting the cell tissue lysates 1 1 in the assay buffer provided in the intracellular kit is recommended Consequently all samples need to be brought to a protein concentration of 0 8 ug L in lysis buffer Take 30 uL of each lysate sample and add it to 30 uL of assay buffer bringing the final concentration down to 0 4 mg mL Then load 25 uL of diluted samples into each well usually in duplicate Appendix 1 Species Cross reactivity Canine e Four serum or plasma samples were usually run in each kit Exceptions are noted e Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits Panel Name EGF Eotaxin FGF 2 Fractalkine G CSF GM CSF GRO 2 Human Cytokine Chemokine Panel 1 4 2 1 1 1 1 1 1 IFNy IL 10 IL 18 IL 1ra IL 2 IL 3 IL 5 IL 6 1 2 3 2 2 2 1 1 IL 7 IL 8 IL 9 IL 10 IL 12 p40 IL 13 IL 15 IL 17A 1 1 1 2 1 2 2 1 IP 10 MCP 1 MCP 3 MDC 10 1 sCD40L TGFa 1 1 2 2 2 1 2 2 TNFa TNFB VEGF A PDGF AB BB 0 2 1 B Panel Name SDF 1o B EOTAXIN 3 CTACK IL 23 TPO TSLP IL 33 Human Cytokine Chemokine Panel 2 1 1 1
44. e Protease Inhibitor Cocktail Roche Applied Science Indianapolis IN at 4 C Super natant was collected and analyzed for the presence of cytokines using a Luminex instrument with a Mouse Cytokine Panel Merck Millipore as per the manufacturer s protocol MILLIPLEX map Mouse Cytokine Chemokine Magnetic Bead Panel Cat No MCYTOMAG 70K Ear tissue from mice treated with vehicle or R348 120 mg kg were harvested after 6 weeks of treatment and snap frozen in liquid nitrogen Ears were homogenized under liquid nitrogen with radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors centrifuged at 13 000 rpm and the supernatant was collected to generate ear lysates Ear lysates were normalized for equal protein concentration For infectious samples If washing with an automatic plate washer add 30 bleach to the waste bottle before washing aspirating the plate If washing with a hand held magnetic bead separator add 30 bleach to a container capable of catching the wash solution decanted from the plate Then at the end of the assay resuspend the beads in 0 1ml of 4 formaldehyde made in 10mM PBS prepared fresh daily instead of sheath fluid before running the plate in the Luminex9 machine Prolonged incubation in this solution may cause bead aggregation Consequently after agitating the plate for 5 minutes on an orbital plate shaker read the plate immediately Jejunal biopsy specimens were
45. e extracted from the sponges just prior to analysis Each individual sponge was weighed to determine the volume of secretions absorbed into the sponges The sponges were then equilibrated in 300 pL phosphate buffered saline PBS 0 25 M NaCl with 10 fetal calf serum for 30 minutes at 4 C The secretions were separat ed using a spin x centrifuge filter unit Costar Cambridge MA centrifuged at 12 000 x g rpm for 20 minutes A dilution factor for the final extract was determined based on the following formula dilution factor x 0 0625 mL 0 3 mL buffer x 0 0625 where x equals the volume of material collected and 0 06 equals the weight of the dry spear 9 Note The weight of the dry sponge is dependent on the lot number Each lot must be weighed This dilution factor was used to calculate the final units of specific antibody and total immunoglobulin measured Normal and cancer tissue specimen weights were determined before protein extraction with Tissue Protein Extraction Reagent T PER Pierce Rockford USA as recommended by the manufacturer Briefly 20 mL of P TER was added to 1 g of tissue and homog enized Samples were centrifuged at 10 000 x g for 5 minutes and the supernatant protein extract was stored at 80 C until cytokine chemokine profiling Skin ear biopsies were pooled from four test animals Biopsies were minced and then repeatedly homogenized with beads in phosphate buffered saline PBS plus Complet
46. el 3 GM CSF TGFa G CSF IFNy IL 2 IL 15 sCD40L IL 17A Non Human Primate Cytokine 1 4 2 2 4 4 1 4 Chemokine IL 1ra IL 13 IL 1B IL 4 IL 5 IL 6 IL 8 10 4 1 4 4 1 4 4 4 IL TNFa 1 12 23 p40 VEGF A IL 18 2 1 1 3 4 Panel Name G CSF Eotaxin GM CSF IL 1 Leptin 10 IL 4 IL 1B Rat Cytokine Chemokine 1 1 1 2 2 2 2 p IL 2 IL 6 EGF IL 13 IL 10 IL 12 p70 IFNy IL 5 2 2 2 2 2 2 2 1 IL 17A IL 18 MCP 1 IP 10 GROo KC VEGF A Fractalkine LIX 1 2 2 2 2 2 2 2 MIP 2 TNFa RANTES 2 2 2 GM CSF IFNy IL 2 IL 6 IL 7 IL 8 IL 15 IP 10 Canine Cytokine Chemokine 3 4 4 4 4 2 4 4 KC IL 10 IL 18 MCP 1 TNFa 3 2 4 4 2 36 CVD kits Panel Name CK MB FABP3 Troponin I Human CVD1 4 1 1 2 Panel Name Macroglobulin Fetuin A AGP Fibrinogen sL Selectin SAP Haptoglobin Human CVD3 4 1 4 4 2 9 Platelet Factor von Willebrand 4 Factor 4 3 Panel Name sE Selectin dPAPP A sPECAM 1 Pentraxin 3 Tissue Factor Troponin T Human CVD4 1 4 4 4 Angiogenesis kits 6 samples run Panel Name EGF ANGPT 2 ET 1 FGF 1 Follistatin IL 8 HB EGF Human 1 3 B 3 9 Angiogenesis Growth Factor VEGF D VEGF A 2 3 Porcine e Four serum or plasma samples were usually run in each kit Exceptions are noted e Data below are the number of samples that showed signal above background For more information contact Technical Sup
47. emokine Panel 1 4 4 1 4 4 4 4 2 IL 10 IL 1B IL 1ra IL 3 IL 4 IL 5 IL 6 IL 7 4 4 4 4 4 4 4 4 IL 8 IL 9 IL 10 IL 12 p40 1L 12 p70 IL 13 IL 15 IL 17A 4 4 4 4 1 4 4 1 IP 10 MCP 1 MCP 3 MDC 10 1 sCD40L TGFa 1 1 4 3 4 1 4 4 TNFa VEGF A PDGF AB BB 0 1 4 Panel Name MCP 4 10 IL 16 MIP 18 6Ckine CTACK IL 23 LIF Human Cytokine Chemokine Panel 2 1 2 9 2 2 9 3 1 TPO TSLP IL 28A IL 33 3 2 2 2 Panel Name IL 17F GM CSF IFNy IL 10 MIP 30 IL 13 IL 15 IL 17A Human TH17 2 2 4 4 1 1 2 4 11 22 IL 9 1 1 IL 33 IL 2 IL 21 IL 4 IL 23 1 8 4 3 4 1 1 2 IL 5 IL 6 IL 17E 11 27 IL 31 TNFa TNFB IL 28A 1 1 1 7 1 1 4 1 35 Cytokine kits continued Granzyme Granzyme Panel Name GM CSF sCD137 IFNy IL 10 A IL 13 B IL 2 Human CD8 2 1 4 1 2 S 1 4 IL 4 IL 5 IL 6 MIP 10 MIP 1p TNFo Perforin 1 1 2 3 1 1 1 Panel Name Eotaxin G CSF GM CSF IFNy IL 10 M CSF IL 1B IL 2 Mouse Cytokine Chemokine Panel 1 4 4 4 4 4 4 4 4 IL 3 IL 4 IL 5 IL 6 IL 7 IL 10 IL 12 p40 IL 13 4 4 4 4 4 4 4 4 IL 15 IL 17A IP 10 MIP 2 KC LIF LIX MCP 1 4 4 4 4 4 4 4 4 10 1 MIG RANTES TNFa IL 12 p70 VEGF A IL 9 4 4 4 4 4 4 4 4 Panel Name EPO Exodus 2 Fractalkine IL 16 IL 21 IL 22 IL 17E IL 28B Mouse Cytokine Chemokine Panel 2 MCP 5 MIP 30 MIP 38 TARC Panel Name MDC IL 23 IL 27 TIMP 1 IL 20 IL 33 Mouse Cytokine Chemokine Pan
48. ere mechanically homogenized with a pestle followed by centrifugation at 4 C Supernatant was transferred to another tube and frozen on dry ice Add Merck Millipore protease inhibitor cocktail at 1 500 to saliva Centrifuge at 10K rpm 10 minutes and dilute supernatant 1 2 with assay buffer prior to assay setup This method significantly improves recovery and reduces bead aggregation Run assay with assay buffer as matrix in standard curve Use an overnight option if available Gut 2009 Apr 58 4 520 9 doi 10 1136 gut 2008 158824 Epub 2008 Nov 20 PMID 19022917 nvest Dermatol 2010 pr 130 4 1023 33 doi 0 1038 jid 2009 358 Epub 009 Nov 12 MID 19907432 gt UN J Immunol 2009 183 3 2183 92 PMID 19596999 Infect Immun 2007 Jan 75 1 481 7 Epub 2006 Oct 16 PMID 17043107 Circ Res 1976 Nov 39 5 659 65 PMID 184975 Sample Type Species Kit Run Procedure Reference if available Skin Extracts Human Human Quantitation of multiple proteins in extracts of D Squame tape samples of human Int J Dermatol 2011 Cytokine Panel 1 scalp using a multiplex skinMAP multiple analyte profile immunoassay D Squame Jan 50 1 102 13 ape strip samples of human scalp skin were extracted with PBS containing 0 2 505 doi 10 1111 j 1365 and 0 5 propylene glycol PG for 30 minutes with sonication on ice The extracts 4632 2010 04629 x were then centrifuged for 5 minutes at 2 100 x g to remove skin so
49. ex Corporation For technical support on all Luminex system or xMAP technology visit www merckmillipore com Imx contact or contact Luminex Corporation Technical Support Phone 512 381 4397 Toll free 1 877 785 2323 Fax 512 219 5114 Email support 2luminexcorp com BioTek All Regions www merckmillipore com biotek contact Technical Support North America 800 242 4685 Outside the U S A 802 655 4740 Email TAC biotek com Merck Millipore Custom Assay Development Need to develop a specific sensitive analytically validated assay for your laboratory We develop reliable custom multiplexed assays using Luminex xMAP technology as well as assays for detecting single proteins ELISAs RIAs and GyroMark HT Assay kits providing you with Reagents immunogen design and antibody development Assay development Manufacturing commercial kits for research use only Contact your Sales Specialist or email us at customassay Q merckmillipore com Is there an analyte panel or species you would like to see in our portfolio biomarkerwish merckmillipore com 47 48 Notes Notes www merckmillipore com Bre Acus ALS MERCK MILLIPORE To place an order or receive technical assistance In Europe please call Customer Service France 0825 045 645 Germany 069 86798021 Italy 848 845 645 Spain 901 516 645 Option 1 Switzerland 0848 645 645 United Kingdom 0870 900 4645 For other
50. fixed with formalin or embedded in optimal cutting temperature OCT compound and snap frozen in liquid nitrogen Protein extracts were prepared from jejunal biopsies embedded in OCT compound by washing them twice with a phosphate buffered saline lysis buffer containing 0 05 sodium azide 0 5 Triton X 100 1 mM phenylmethylsulfonyl fluoride and protease inhibitors Complete Mini protease inhibitor cocktail Roche Diagnostics Indianapolis IN After OCT compound removal the tissues were minced in 1 mL of lysis buffer with a sterile disposable homogenizer on ice for 5 minutes The homogenates from the tissues were then sonicated for 1 minute on ice After centrifugation at 10 000 x g for 15 minutes the supernatant was collected and stored at 80 C or immediately as sayed to determine the protein concentration with a bicinchoninic acid protein assay kit Pierce Rockford IL For lipemic and plasma samples the blood needs to be collected on ice centrifuged in a refrigerated centrifuge aliquoted and frozen at 20 C for short term lt 2 months and 70 C for long term Prior to assay setup thaw samples and centrifuge a 10 000 rpm for 5 minutes Spool off the lipid layer from the surface using a cotton swab and use the supernatant below lipid layer for the assay Footpad popliteal lymph nodes from mouse subjects were harvested combined and placed in 200 uL of PBS containing 1 x protease inhibitors Roche The lymph nodes w
51. g per well 10 protein in the final 25 uL that is loaded into each assay well is recommended Working backwards from there 10 ug 25 uL 0 4 ug uL mg ml Diluting the cell tissue lysates 1 1 in the assay buffer cytoplasmic proteins Partially ionic detergents Triton X 100 are recommended in lysis buffers for cytoplasmic or n provided in the intracellular kit is recommended membrane bound proteins C Consequently all samples need to be brought lonic detergents sodium dodecyl sulfate 5 Y 4 3 tei tration of 0 8 Lin lysis buffer SDS are recommended in lysis buffers for Mol insist Take 30 uL of each lysate sample and add it to 30 uL of membrane bound nuclear or mitochondrial mE assay buffer bringing the final concentration down to 0 4 mg mL Then load 25 uL of diluted samples into each well usually proteins If using SDS in the lysis buffer i e Radioimmunoprecipitation assay RIPA buffer then cell lysate must be diluted to less than in duplicate 0 05 SDS for assays to detect intracellular The Direct Detect spectrometer enables you to determine protein sample concentrations quickly and accurately For more information visit www merckmillipore com directdetect proteins such as cell signaling proteins gt w aie gt 19 lt Preparation of Reagents General
52. he best one e f samples fall outside the dynamic range of the assay dilute the samples further with the appropriate matrices media and repeat the assay How Merck Millipore Monitors Avoids Lot to lot Drift Lot to lot drift is monitored and mitigated using full curve comparison and comparing the relative potency of each analyte against a reference lot e All data are compiled in a single database and trend charts are maintained in our records MILLIPLEX map standard points maintain consistent values from lot to lot unlike other kits that may have values that vary lot to lot Comparison of MILLIPLEX Analyst 5 1 StatLlA and Bio Plex Analysis Software Analyte IFNy Kit Rat Cytokine Chemokine Units pg mL MILLIPLEX Analyst 5 1 StatLIA Bio Plex Standard1 14 7 3 0 Standard2 57 4 67 0 53 7 Standard3 241 6 245 0 248 6 Standard4 932 1 897 0 908 1 Standard5 3683 0 3858 0 3820 0 Standard6 15184 0 14769 0 14824 5 Standard7 59874 0 61392 0 60975 2 Unknown1 3401 1 0e 07 OOR lt Unknown2 16 8 8 0 OOR lt Unknown3 51 3 60 0 45 0 Unknown4 197 1 205 0 205 8 Unknown5 844 1 809 0 821 3 Unknown6 3412 0 3564 0 3531 3 Unknown7 14639 0 14296 0 14339 3 Unknown8 70718 0 82002 0 78697 5 Unknown9 3401 1 0e 07 OOR lt Unknown10 3401 1 0e 07 OOR Unknown11 3401 1 0e 0
53. hemokine 1 2 2 4 3 4 2 1 IL 13 IL 10 IL 12 p70 IL 18 MCP 1 IP 10 GROo KC VEGF A 2 4 1 1 3 2 2 2 LIX MIP 2 TNFa RANTES 4 4 2 2 Panel Name GM CSF IL 15 IP 10 IL 18 Canine Cytokine Chemokine 4 1 4 2 GM CSF IL 1B IL 1ra IL 2 IL 4 IL 6 IL 10 IL 12 Porcine Cytokine Chemokine 3 4 1 2 3 1 1 2 IL 18 2 CVD kits Panel Name ADAMTS13 D Dimer FABP5 GDF 15 Myoglobin sP Selectin Human CVD2 4 3 4 4 9 1 Panel Name AGP Haptoglobin von Willebrand Factor Human CVD3 2 4 4 Panel Name MMP 9 PAI 1 total sP Selectin Mouse CVD1 2 3 3 Angiogenesis kits 6 samples run Panel Name ANGPT 2 BMP 9 FGF 1 Follistatin HB EGF Human Angiogenesis Growth Factor 5 4 1 5 5 33 Hamster Four serum or plasma samples were usually run in each kit Exceptions are noted Data below are the number of samples that showed signal above background For more information contact Technical Support Cytokine kits PDGF AB Panel Name EGF FGF 2 Fractalkine IL 1 IL 3 MIP 18 PDGF AA BB Human Cytokine Chemokine Panel 1 2 1 3 1 4 1 1 Panel Name SDF 10 8 CTACK IL 21 Human Cytokine Chemokine Panel 2 4 1 1 Panel Name GM CSF IL 13 17 4 1 4 4 Panel Name G CSF GM CSF IFNy IL 10 M CSF IL 7 IL 12 p40 IL 13 Mouse Cytokine Chemokine Panel 1 1 1 2 1 1 1 1 4 IL 15 IL 17A IP 10 LIX MIP 10 MIP 18 MIG RANTES 2 1 1 1 1 2 2
54. ix found only in MILLIPLEX kits that is added to the standard wells to mimic the environment in which native analytes are present in serum plasma The selected matrix most often consists of serum plasma pool with all endogenous and cross reacting proteins extracted Sheath fluid Luminex catalog number 40 50000 Luminex Sheath Fluid is intended for use as the delivery medium of the sample to the optics component of the Luminex 100 200 system and the FLEXMAP 3D system Spike recovery Data representing mean percent recovery of spiked standards ranging from low medium and high concentrations in serum matrices for a defined number of samples see Accuracy Stability Resistance or the degree of resistance to chemical change or disintegration Standard curve Calibration curve A graphic plot of median fluorescence intensity versus the known concentration of test substances in a set of standards usually prepared by serial dilution or incremental addition xMAP _ Multi Analyte Profiling technology Flexible open architecture design that can be configured to perform a wide variety of bioassays developed by the Luminex Corporation xPONENT software Luminex acquisition software with analysis capabilities Sources include thefreedictionary com merriam webster com luminexcorp com scientistsolutions com regulatory com dictionary com and MILLIPLEX protocol More Resources for Technical Support Lumin
55. lids that might PMID 21182510 interfere in the assay Subsequently the extracts of D Squame tape samples were ransferred into 96 well polypropylene deep well plates and frozen at 80 C for Human Skin Panel Cat No SKINMAG 50K and soluble protein analyses as previously reported Tears Human Human Polyurethane minisponges were obtained commercially PeleTim VOCO GmbH Cux Cytokines haven Germany Specialized nurses carried out the procedures A single polyurethane minisponge was laid on the outer third of the lower eyelid margin After 5 minutes of ear collection the sponge was recovered and placed in the narrow end of a truncated Gilson micropipette tip adapted to a 1 5 mL tube Eppendorf Fremont CA and centrifuged at 6 000 rpm for 5 minutes Tear samples from both eyes were pooled and immediately stored at 80 C until they were used for the immunoassay Tears Human Tear collection was performed before any other test and with a minimum of Mol Vis 2010 May 19 16 862 10 minutes after the patient answered the two symptom questionnaires Unstimulated 73 tear samples were collected non traumatically from the external canthus of open PMID 20508732 eyes avoiding additional tear reflex as much as possible Glass capillary micropipettes Drummond Broomall PA were used to collect 1 uL of tears Each sample was then diluted 1 10 in a sterile collection tube containing ice cold Cytokine Assay Buffer Merck Millipore Tubes with
56. nical Support To determine cross reactivity for other species for a panel or analyte See the Species Cross reactivity Tables in Appendix 1 For intracellular assay kits we analytically validate the assay with human cell tissue culture samples However we provide the species homology for each analyte in a table on the product detail page on our website We have also compiled a list of this information on our website Kits Kit Species Cross Reactivity www merckmillipore com kits species MAPmate assays MAPmate Species Cross Reactivity amp Buffer Table www merckmillipore com mapmates sp Search the latest edition of the Analyte Quarterly www merckmillipore com milliplex 3 General Assay Information All kits are for Research Use Only How to design a customized kit Select your panel of interest for example Always read the entire protocol before proceeding Human Cytokine Chemokine Panel 1 555644864444 ee ee ee nh rn hn Cat No 6 For questions issues with Luminex instruments Choose only the analytes you want from that contact Luminex at 7 panel for example you may need only five All Regions analytes IL 2 IL 6 IL 10 GM CSF VEGF A www merckmillipore com Imx contact gt an lt s D c y fe Add the number of analytes you chose to the Technical Support catalog number HCYTOMA
57. nstrument that combines differentially dyed fluorescent microsphere sets with an innovative instrument design to enable precise rapid multiplexing of up to 500 unique assays within a single sample Other features include an automated probe height adjustment simplified routine maintenance operations and an intuitive software interface Inter assay precision Precision generated across two different concentrations of analytes across a defined number of different assays Intra assay precision CV Precision generated across two different concentrations of analytes in a single assay Linearity The ability within a given range to obtain test results which are directly proportional to the concentration amount of analyte in the sample Luminex 200 system A Luminex instrument that provides a complete mid to high range solution for rapid accurate biomarker quantification The Luminex XY Platform Luminex XYP complements this instrument system by automating the sequential positioning of each well of a microtiter plate Magnetic beads MagPlex9 Similar to MicroPlex9 microspheres MagPlex microspheres are carboxylated polystyrene micro particles or beads that have been dyed into spectrally distinct sets or regions allowing them to be individually identified by a Luminex instrument These uniquely coded beads provide a user an addressable substrate on which to perform multiple bio analytical
58. om or Use appropriate pipetting technique refrigerator with power access for the orbital Hold the pipette at the same angle each time shaker e Use pipettes calibrated for values in the middle nese range not extremes When running samples in duplicate a maximum of 38 samples can run per kit e Warm reagents to room temperature 20 25 C before mixing For assays requiring overnight incubation in a cold room warm reagents to room temperature on the second day as well 20 Ke lt gt lt 1 2 3 4 5 6 7 8 9 10 11 12 Standard 0 QC 2 A Background Standard 4 Control Standard 0 QC 2 8 Background Standard 4 Control Standard 1 Standard 5 Sample 1 Standard 1 Standard 5 Sample 1 E Standard 2 Standard 6 Sample 2 F Standard 2 Standard 6 Sample 2 QC 1 G Standard 3 Control Etc QC 1 H Standard 3 Control 96 Well Plate Map Sample map showing placement of standards QCs background and samples e pre wet the plate use 150 uL wash buffer or e f the detection antibody has been accidentally assay buffer aspirated off or poured off before adding SAPE to rcc RR e the well it is possible to recover the assay Add 20 ul to 50 uL of detection antibody and continue to follow the protocol f you accidentally use wash buffer instead of assay
59. on e Longer expiration dates can be requested Please contact your Sales Specialist General Information 4 Materials Required But Not Provided e Adjustable pipettes with tips capable of delivering Before you open a MILLIPLEX kit check your 25 uL to 1000 uL instrument saesisessecisesssessiessecsiesesosissesossssesosssoesosssecsse e s the instrument calibrated Multichannel pipettes capable of delivering 5 uL to Has it been maintained 0 TAROT e Bead washer either automated or manual Laboratory vortex mixer e Automated magnetic bead plate washers 0 1 1 1 1 1 10 7 BioTek 405 LS Magnetic 96 2311 Washer e Water bath sonicator Branson Ultrasonic Cleaner Cat No 40 0944 Model B200 or equivalent BioTek 405 LS Magnetic Vacuum Filtration 8 Sonicator probes not recommended 96 well Washer Cat No 40 095 bra 405 TS Magnetic 96 well Washer Orbital titer plate shaker Lab Line Instruments Complete with Touch Screen and Ultrasonic Model 4625 or equivalent Cleaning Cat No 40 096 Very important For incubating assays overnight BioTek 405 TS Magnetic Vacuum a power supply must be available for the orbital Filtration 96 well Washer Complete with shaker in refrigerator or cold room Touch Screen and Ultrasonic Cleaning
60. port Cytokine kits Panel Name EGF Eotaxin FGF 2 Fractalkine GM CSF GRO IFNo2 IFNy Human Cytokine Chemokine Panel 1 1 2 4 9 9 9 3 2 IL 10 IL 1 IL 1ra IL 4 IL 5 IL 6 IL 7 IL 8 3 4 4 3 2 2 3 3 IL 9 IL 10 IL 12 p40 1L 12 p70 IL 13 IL 15 IL 17A MCP 3 3 4 4 0 3 3 2 3 MDC MIP 10 1 sCD40L TGFa TNFB VEGF A RANTES 3 4 3 4 4 4 1 1 PDGF AB BB 4 Panel Name SDF 10 8 IL 16 IL 23 TPO IL 20 IL 28A IL 33 Human Cytokine Chemokine Panel 2 4 1 2 1 1 1 1 Panel Name IL 17F GM CSF IL 10 IL 15 IL 22 IL 9 IL 1B IL 33 Human TH17 2 3 1 3 4 3 3 3 IL 2 IL 4 IL 23 IL 17E IL 27 IL 31 TNFB IL 28A 4 1 3 2 2 3 2 1 Granzyme Granzyme Panel Name GM CSF sCD137 IFNy IL 10 A IL 13 B IL 2 Human CD8 2 2 2 1 2 9 2 2 IL 4 IL 5 IL 6 sFasL MIP 10 1 TNFa Perforin 2 2 2 2 2 2 2 2 37 Cytokine kits continued Panel Name GM CSF IFNy IL 10 IL 13 IL 17A IL 1B IL 2 Human High Sensitivity T Cell 1 1 2 1 2 3 2 2 IL 4 IL 23 IL 5 IL 6 IL 8 MIP10 D 3 1 1 4 1 1 Panel Name IL 17E GM CSF IFNy MIP 3o IL 1B IL 2 IL 4 IL 5 Mouse TH17 2 3 2 2 2 3 2 3 IL 6 IL 21 IL 22 IL 28B IL 10 IL 23 IL 12 p70 11 27 2 4 4 3 3 S S 2 2 4 13 1 3 2 4 2 4 4 2 4 2 sCD40L 4 Panel Name GM CSF TGFo G CSF IFNy IL 2 IL 10 IL 15 sCD40L Non Human Primate Cytokine 0 4 2 3 4 0 4 1 Chemokine IL 17A IL 1ra 1 13 IL
61. r up down strokes at Setting No 3 using a Polytron Brinkmann Instruments Inc Westoury NY he crude homogenate was centrifuged at 3 000 x g for 15 minutes and the fat cake was discarded The infranate was made up to 1 vol vol Iriton X 100 was used to solubilize enzymes from the particulate compart ment into the tissue homogenate The supernatant resulting from centrifugation at 15 000 x g for 20 minutes at 4 C was stored in aliquots at 80 C Adipose biopsies 50 75 mg were homogenized on ice in 1 mL of the kit assay buf fer 10 mmol L PBS 0 08 wt vol sodium azide 1 wt vol BSA pH 7 4 The homogenate was further diluted 25 fold in assay buffer to minimize assay interfer ences 10 uL of dilute homogenate was incubated in 96 well plate with 25 uL of capture antibody conjugated beads and 65 uL assay buffer for 1 hour ambient Beads were washed 10 mmol L PBS 0 05 vol vol Proclin 0 05 vol vol Tween 20 pH 7 4 and 50 biotinylated detection antibody cocktail added for 30 minutes ambient followed by further washing and incubation with 50 uL streptavidin phycoerythrin for 30 minutes at ambient temperature After final washing beads were resuspended in 150 uL Luminex sheath fluid for analysis Approximately 100 200 mg adipose tissue SAT and VAT from each subject was homogenized in 250 uL of ice cold homogenization buffer The homogenate was centrifuged at 3 000 x g for 15 minutes at 4 C
62. reactions in a single well MAGPIX system A Luminex instrument based on CCD imaging technology which allows for a compact more robust system Streamlined startup and shutdown protocols and minimal maintenance requirements make the system easy to operate and maintain MFI Median fluorescence intensity MILLIPLEX 5 1 Analyst software Merck Millipore analysis software package that is able to automatically import data from Luminex instruments providing better data from the low and high ends of standard curves and comprehensive detailed reports and enhanced visualization MILLIPLEX A broad portfolio of multiplex immunoassays that includes immunology metabolism cardiovascular disease cancer neuroscience toxicity cellular metabolism and cell signaling pathways for a variety of species MinDC minimum detectable concentration The lowest concentration at which an analyte can be reliably detected Precision Generated from the mean of the CVs from a defined number of reportable results Premixed kit A MILLIPLEX kit in which the capture beads have been mixed together There is often an option of choosing either a premixed kit or a configurable kit e g FCYTOMAG 20K PMX Feline Cytokine Chemokine 19 plex Premix Quality controls QCs Medium low and medium high standard points used to qualify assay performance SAPE Streptavidin phycoerythrin Serum matrix An appropriately selected matr
63. s Required but Not Provided Sample Collection and Preparation Preparation of Reagents Immunoassay Procedure Plate Washing Equipment Settings MILLIPLEX Analyst 5 1 Software Intracellular Assays Appendices 1 Species Cross reactivity 2 Sample Preparation 3 Other Sample Types 4 Cell Signaling MAPmate Buffer Table Figure 1 Two different fluorescence detection methods for acquiring and analyzing bead based assay data Introduction The Luminex xMAP Technology MILLIPLEX map is based on the Luminex xMAP bead based assay platform one of the fastest growing and most respected multiplex technologies supporting applications throughout the life sciences This platform is capable of performing a variety of bioassays including immunoassays on the surface of fluorescent coded magnetic MagPlex and nonmagnetic MicroPlex bead microspheres Luminex uses proprietary techniques to internally color code microspheres with multiple fluorescent dyes Through precise concentrations of these dyes distinctly colored bead sets of 500 5 6 um non magnetic or 80 6 45 magnetic polystyrene microspheres can be created each of which is coated with a specific capture antibody After the target protein from a test sample is captured by the bead a biotinylated detection antibody is introduced The reaction mixture is then incubated with Streptavidin PE conjugate
64. sistent and accurate data especially if multiple users will be generating data in collaboration Improper or inconsistent technique can affect delivery volumes and impact data reliability Training or best practices for pipetting including that pipettors are properly calibrated can substantially increase pipetting precision For tips on proper pipetting techniques visit www merckmillipore com mlo Before you collect samples to run an assay it s important to read the entire protocol If you have any questions contact Technical Support or your Sales Specialist e If the protocol states that the kit can be used in either serum or plasma and you have the option choose serum because it tends to be cleaner Beconsistent with the use of sample types within a study project f you are trying to decide whether to collect serum or plasma samples ask yourself what you have observed from preliminary data publications or collaborators Still unsure Contact Technical Support Freeze thaw limits Multiple freeze thaw cycles may reduce the stability of the analytes Vortexing samples and beads To get a homogeneous prep sample bead vortexing is recommended especially after the sample has been centrifuged and the supernatant separated Tips on using tissue culture media as assay buffer e f the medium used to grow the cells is used as the matrix take care that there are no proteases or any supplemen
65. t Total 46 620MAG H e c Met HGFR pan Tyr 46 651MAG H c Met HGFR Total 46 650MAG H e e CREB Ser133 46 631MAG H M R e e CREB Total 46 632MAG EGF Receptor Tyr 46 603MAG H e EGF Receptor Total 46 606MAG H e e ERK MAPK 1 2 Thr185 Tyr187 46 602MAG H M R ERK MAPK 1 2 Total 46 609MAG e e GSK3B Ser9 46 690MAG e e GSK3 Total 46 689MAG H2A X Ser139 46 692MAG H M e e HIF 10 Total 46 665MAG e e HSP27 Ser78 46 607MAG H e e HSP27 Total 46 608MAG H e IRS1 pan Tyr 46 627MAG H M R e IRS1 Total 46 628MAG e Ser32 46 643MAG H e e IxBo Total 46 644MAG H e e JNK SAPK1 Thr183 Tyr185 46 613MAG HM e e JNK SAPK1 Total 46 618MAG HM e Lck Tyr394 46 712MAG H M R e e MEK1 Ser222 46 670MAG e e MEK1 Total 46 669MAG HM e e mTOR Ser2448 46 686MAG e e mTOR Total 46 685MAG H M R e NF B Ser536 46 702MAG H e e NF B Total 46 701MAG H e e p21 Total 46 621MAG H e 38 5 2 Thr180 Tyr182 46 610MAG H M R e 38 5 2 Total 46 612MAG p53 Ser15 46 663MAG H e e p53 Total 46 662MAG H e 7056 Thr389 412 46 629MAG H M R e 7056 Total 46 630 e o Cleaved PARP Total 46 656MAG H e 5 380 46 679MAG e e PTEN Total 46 678MAG H M R 56 Ser235 Ser236 46 714MA
66. tear samples were kept cold 4 C during collection and stored at 80 C until assayed Cell or Tissue Protocol varies depending on tissue types and or analytes of interest Generally most Extraction protocols that are used in ELISAs can be used but here are some guidelines in selecting a method 1 Homogenize cells or tissues mechanically eg ultrasonication in a PBS based buffer containing protease inhibitors like aprotinin or an inhibitor cocktail and low 0 2 non ionic detergent concentration 2 Extraction medium should not contain any organic solvents like DMSO etc 3 Centrifuge the extract and freeze supernatant at 20 C 4 Use the extraction medium as matrix in blank standard curve and QCs Tumor Mouse Mouse Tumors were treated with DMXAA After harvesting at 6 24 and 48 hours the tumors Cancer Res 2005 Dec Homogenates Cytokines were sonicated for 30 seconds in 1 mL of complete buffer 50 mL PBS containing 15 65 24 11752 61 one tablet of antiprotease cocktail Roche Indianapolis IN Tissues were then spun PMID 16357188 at 3 000 rpm for 10 minutes and filtered through a 1 2 um syringe filter unit Total protein in each sample was determined Urine Typically measurement of analytes in urine requires either a 24 hours urine collection or second morning void collection For the second morning void urine the analyte value is normalized against creatinine i e the analyte is expressed as units mg of crea
67. tinine Mix urine samples 1 1 with assay buffer and incubate on the plate ap proximately 20 minutes on a shaker prior to addition of the beads Use assay buffer as matrix for standard curve controls and blank The assumption is that this helps neutralize the sample thereby improving recovery While these alternate methods have been tried by Merck Millipore or our end users using MILLIPLEX map kits we cannot guarantee methods will work with all samples These procedures have not been analytically validated Appendix 4 Cell Signaling Buffer Table Species Cross Reactivity and Buffer Compatibility Magnetic Bead MAPmate Kits Cat No Species Homology AB1 AB2 Total 46 713MAG H M R GAPDH Total 46 667MAG H Akt PKB assay buffer 1 5 473 46 601MAG H M R Akt PKB assay buffer 1 Thr308 46 645MAG H M R e Akt PKB assay buffer 1 Total 46 605MAG H M R Akt PKB assay buffer 2 Ser473 46 677MAG H M R Akt PKB assay buffer 2 Total 46 675MAG H M R 112 46 694MAG H M e e BAD Total 46 695MAG HM e Caspase 3 Active 46 604MAG I c Jun Ser73 46 622MAG H M R c Kit pan 46 619MAG H c Ki
68. tracts use the culture or extraction medium as the matrix solution in the blank standard curves and controls e f samples are diluted in assay buffer use the assay buffer as the matrix For cell tissue homogenates the final cell or tissue homogenate should be prepared in a buffer that NOTE to solubilize nuclear mitochondrial proteins you must use either SDS or another method such as ultrasonication to puncture the tough nuclear mitochondrial membranes Reducing agents like B mercaptoethanol or dithiothreitol are not recommended For more information about the compatibility of buffers with MILLIPLEX9 map Signaling kits contact Technical Support has a neutral pH contains minimal detergents Perform all dilutions with lysis buffer not assay or strongly denaturing agents and has an ionic buffer or phosphate buffered saline PBS strength close to physiological concentrations Avoid debris lipids and cell tissue aggregates Table 1 Detergent compatibility with MILLIPLEX intracellular assays Centrifuge samples before use Maximum allowed protein MILLIPLEX assay Type of detergents Protein localization concentration compatibility Preparation of Cell Lysates for Non ionic detergents Cytoplasm 5 mg mL Yes Intracellular Assays 96 well Plates Partially ionic Cytoplasm S mafil Yes detergents Membrane bound Membrane bound Nucleus Mitochondria e For adherent cell lines seed 40 00
69. ts that may interfere with the assay or generate inaccurate results e g cytokines human serum etc Some kits for metabolism biomarkers require adding protease inhibitors to samples Others may require a sample extraction or acidification Consult the protocol of the appropriate kit See Sample Preparation outlines for kits requiring sample extraction acidification etc in Appendix 2 Preparation of Serum Plasma Samples For serum or plasma samples that require a dilution instead of Neat use the serum matrix provided in the kit as the diluents Hemolysis can result in increased proteolytic activity and analyte degradation primarily due to enzymes released from lysed cells Trace hemolysis in samples collected with protease inhibitors may be acceptable but gross hemolysis will probably interfere with assay performance Hemoglobin at gt 10 mg ml is known to interfere with antigen antibody interactions If you want to run a MILLIPLEX kit using samples other than serum or plasma we have protocols to address tissue lysates urine blood spots gingival fluid nasal lavage fluid tears cerebrospinal fluid CSF bronchoalveolar fluid saliva and cervical vaginal secretions as well as protocols that are modified for use with small volume samples Please contact Technical Support or refer to Appendix 3 Preparation of Tissue Culture Supernatants For cell culture supernatants or tissue ex

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