Bio-Plex Pro™ Cell Signaling Assays - Bio-Rad
Contents
1. 5 23 5 236 74 HER 2 Tyr 30 Smad2 Ser Ser e7 14 HSP27 Ser 51 Src Tyr 9 42 IGF IR 43 Stat Tyr 61 IR b Tyr 9 43 Stat3 Ser 52 IRS 1 Ser 5 Ser9 9 76 Stat3 Tyr 52 IkB a Ser Ser 67 Syk Tyr 65 JNK Thr Tyr 34 VEGFR 2 Tyr 29 Lyn Tyr 33 ZAP 70 Tyr S 64 MEK1 Ser Ser 27 Total Targets Bead Region Total Targets Bead Region Akt wa JNK 34 Btk 39 MEK1 27 c Jun 56 mTOR 46 CREB 19 p38 MAPK 36 Erk 1 2 38 p70 S6 Kinase 55 GSK3 18 PTEN 22 HER 2 30 Smad2 14 IGF 1R 43 Src 42 IkBa 67 ZAP 70 64 Housekeeping Proteins Bead Region Housekeeping Proteins Bead Region Human GAPDH 21 p Actin 47 9 Click Enter Controls Info and for user specified controls select an analyte from the dropdown menu then enter a description and concentration Repeat for each additional analyte in the assay For Bio Rad cell lysate controls format the appropriate wells as controls enter descriptions but leave the concentrations blank Alternatively both blanks and controls can be formatted as samples with clear descriptions 24 Ed Bio Ples ager D Prot 19 File Edit View Format Options Window Help n s 5 21 Lsm Protocol Settings Plate Formatting Plate Groupings 3 N s o s e 2o s o 2s oo ESI Es Ex ESI ES E Es e To n i s oo 2 e 2o 22 s o e o 2e o 22 ps En2. 25 excess to ensure enough volume ul x 0 25 ul 15 16 c Total volume of 100x streptavidin PE ul ul ul 15 16 17 d Volume of 100x streptavidin PE required yl 100 ul 17 18 e Volume of detection antibody diluent required ul ul ul 17 18 19 33 Safety Considerations Eye protection and gloves are recommended while using this product Consult the MSDS for additional information Human source material Treat as potentially infectious The lysates provided with Bio Plex Pro cell signaling assays contain components of human origin The components are Known to contain an agent that requires handling at Biosafety Level 2 containment as defined by U S government publication Biosafety in Microbiological and Biomedical Laboratories Centers for Disease Control 1999 These agents have been associated with human disease These components have not been screened for hepatitis B human immunodeficiency viruses or other adventitious agents Handle Bio Plex phosphoprotein positive and negative controls as potentially biohazardous material under at least Biosafety Level 2 containment Legal Notices Acrodisc and Supor are trademarks of Pall Corporation FLEXMAP MagPlex xMAP and xPONENT are trademarks of Luminex Corporation Barnstead and Lab Line are trademarks of Thermo Fisher Scientific Sonifier is a trademark of Branson Ultrasonics Corporation The Bio Plex suspension array system includes fluorescently labeled mi
3. Rad Technical Support for more information on defining editing or importing wash station programs 14 Table 5 Summary of wash steps and settings After each assay incubation step perform the appropriate wash step as shown below Bio Plex Pro Wash Station Handheld Magnet Vacuum Manifold Wash Program Settings and Manual Wash Steps Assay Step of Washes Volume ul Magnetic Soak min 1 Add beads to plate 2 200 1 2 Sample incubation 3 200 1 3 Detection Ab incubation 3 200 1 1 4 SA PE incubation 3 200 No magnetic soak for vacuum filtration Setting up a Vacuum Manifold Calibrate the vacuum manifold by placing a standard 96 well flat bottom plate on the unit and adjusting the pressure to between 1 and 3 Hg In general 200 ul liquid should take 5 6 sec to clear the well For detailed instructions refer to bulletin 10005042 Using a Vacuum Manifold After each incubation place the filter plate on a calibrated vacuum apparatus and remove the liquid by vacuum filtration To wash add 200 ul wash buffer to each well and remove the liquid as before Ensure that all wells are exposed to the vacuum Thoroughly blot the bottom of the filter plate with a clean paper towel between each vacuum step to prevent cross contamination Place the assay plate on the plastic plate holder tray as needed Before each incubation gently cover the plate with a new sheet of sealing tape Avoid pressing down over t
4. contact Bio Rad Technical Support The validation kit should be run monthly to ensure optimal performance of fluidics and optics systems Refer to either the software manual or online Help for instructions on how to conduct validation 21 Start Up System Bio Plex 100 200 or similar 1 Empty the waste bottle and fill the sheath fluid bottle before starting if high throughput fluidics HTF are not present This will prevent fluidic system backup and potential data loss 2 Turn on the reader XY platform and HTF if included Allow the System to warm up for 30 min if not already done 3 Select Start up A and follow the instructions If the system is idle for 4 hr without acquiring data the lasers will automatically turn off To reset the 4 hr countdown select Warm up and wait for the lasers optics to reach operational temperature Calibrate System 4 Select Calibrate e and confirm that the default values for CAL 1 and CAL2 are the same as the values printed on the bottle of Bio Plex calibration beads Use the Bio Plex system low RP1 target value even if the assays will be run at high RP1 Bio Plex Manager version 6 1 and higher will automatically calibrate at both high and low RP1 settings although only the low RP1 value option is listed under CAL2 5 Select OK and follow the software prompts for step by step instructions for CAL1 and CAL2 calibration Note In Bio Plex Manager version 6 1 and higher startup warm u
5. settings described below apply to Luminex 100 200 and FLEXMAP 3D or Bio Plex 3D instruments For the Bio Plex MAGPIX reader use the default instrument settings 1 Select MagPlex as the bead type for magnetic beads This automatically sets the DD gates 2 Volume 50 ul 3 Refer to Table 11 to select the appropriate PMT setting for your instrument 4 Plate name 96 well plate 5 Analysis type Qualitative Select Analytes to set up the panel 1 Enter 50 in the Count field 2 Select the bead region and enter the analyte name 3 Click Apply all for Units and Counts Select Stds and Cirls 1 Enter descriptions and other information as applicable After the assay is complete select Results then select Saved Batches 27 Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio Plex Pro assays If you experience any of the problems listed below review the possible causes and solutions provided Suboptimal assay performance may also be due to the Bio Plex suspension array reader To eliminate this possibility use the Bio Plex validation kit to assist in determining if the array reader is functioning properly Table 13 Troubleshooting guide Possible Solutions Problem Possible Causes Low signals for Sample protein concentration experimental too low or too high samples Poor cell lysate quality Low signals for Incorrect dilution of detection exper
6. 36 g PMSF in 5 ml DMSO Store as aliquots at 20 C Add PMSF to the cell lysis buffer at a final concentration of 2 mM Reconstitute cell lysis factor to 100x with 250 ul of diH O and vortex to mix Add the reconstituted factor to the cell lysis buffer to a final 1x working concentration Adherent Cells 1 Stop the treatment reaction by aspirating the culture medium and quickly rinsing the cells with ice cold cell signaling cell wash buffer bottle with the blue cap The volume of buffer required is the same as the volume of aspirated cell culture medium Keep the cells on ice during all steps when possible 2 Completely remove the buffer before lysing the cells 3 Immediately add the cell lysis buffer to the cells The amount of lysing solution needed depends on the cell density in the culture vessel for example add 1 5 2 ml of lysis buffer to a 10 cm dish that is 8096 confluent Note It may be necessary to lyse the samples with different volumes of cell lysis solution to obtain the specified protein concentration range 4 Scrape the cells with a cell scraper collect cell suspension into an appropriately sized tube and gently rock for 20 min at 4 C 5 Perform either of the following to remove insoluble cellular particulates a Centrifuge the cell lysate solution at 4 500 x g for 20 min at 4 C and then filter the lysate using a 0 45 um syringe filter If the lysate volume is not adequate for filtration centrifuge
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8. Bio Plex Pro Cell Signaling Assays Instruction Manual For technical support call your local Bio Rad office or in the U S call 1 800 424 6723 For research use only Not for diagnostic procedures Table of Contents Introduction Principle Kit Components and Storage Recommended Materials Assay Workflow Important Considerations Detailed Instructions 1 Prepare the Samples 2 Plan the Plate Layout 3 Prepare the Wash Method 4 Run the Assay 5 Read the Plate Troubleshooting Guide Plate Layout Template Calculation Worksheet Safety Considerations Legal Notices Ordering Information i on O OC A N 14 16 21 28 31 32 34 34 35 Introduction Bio Plex Pro Cell Signaling Assays Cell signaling is a complex process through which cells receive and respond to stimuli from the surrounding environment For example circulating cytokines and chemokines elicit a response from lymphocytes by binding to cell surface receptors and activating intracellular phosphoprotein signaling cascades This turns on and off specific genes in the nucleus thus regulating protein expression cell growth proliferation motility and survival Chang and Karin 2001 Aberrant signaling can lead to serious pathologies including cancer autoimmune diseases cardiovascular disease and neurological disorders Understanding which cell types and signaling pathways are involved ina disease allows researchers to develop more precisely targ
9. Software The Bio Plex Pro cell signaling assays described in this manual are compatible with all currently available Luminex based life science research instruments Assays can be read and analyzed with either Bio Plex Manager software or Luminex xPONENT software Section 5 Read the Plate under Detailed Instructions Detailed Instructions The following pages provide detailed instructions for each step of the assay procedure including sample preparation running the assay and reading the plate with Bio Plex Manager and Luminex xPONENT software 1 Prepare the Samples Considerations The degree of phosphorylation of a given analyte is highly dependent on the cell type and cell stimulation or treatment conditions a Cell lines may vary in their signaling responses to the same stimulation The suggested final protein concentration range in the assay is 3 200 ug ml 0 15 10 ug per assay well except for PIBK p85 Tyr 5 which is 31 1 000 ug ml 1 6 50 ug well Optimization of cell lysate concentration may be needed based on target protein expression levels a Cell lysate should be clear of particulate matter before use Cell Lysates The Bio Plex Pro cell signaling reagent kit catalog 171 304006M is required for preparing lysates derived from cell culture and tissue samples Just before use prepare an adequate volume of cell lysis buffer by adding PMSF and cell lysis factor QG Prepare 500 mM PMSF by dissolving 0 4
10. bottle 10 ml Resuspension buffer 1 bottle 40 ml Streptavidin PE 100x 1 tube Flat bottom plate 1 plate Sealing tape 1 pack of 4 Assay Quick Guide 1 booklet Bio Rad Cell Lysate Controls optional Positive control treated or untreated 50 ug per vial Negative control phosphatase treated 50 ug per vial Users can mix compatible singleplex sets to create their own multiplex assays Refer to Table 3 to identify the appropriate controls for your phosphoprotein or total target of interest Volumes shown are approximate Storage and Stability Kit contents should be stored at 4 C and never frozen Coupled magnetic beads and streptavidin PE should be stored in the dark All components are guaranteed for a minimum of six months from the date of purchase when stored as specified Table 2 Recommended materials Item Ordering Information Bio Plex Pro Assays Quick Guide 3 Bio Plex 200 system or Luminex system with HTF Bio Plex validation kit Run monthly Bio Plex calibration kit Run daily to standardize fluorescence signal Bio Plex Pro wash station For use with magnetic bead based assays only Bio Plex handheld magnetic washer For magnetic bead based assays only Bio Plex Pro flat bottom plates forty 96 well plates For magnetic separation on the Bio Plex Pro wash station Microtiter plate shaker IKA MTS 2 4 shaker for 2 or 4 microplates or Barnstead Lab Line Model 4625 plate shaker or e
11. by pipetting the required volume into the tube containing wash buffer Vortex Each well of the assay requires 2 5 ul of the 20x stock adjusted to a final volume of 50 ul in wash buffer Note To minimize volume loss use a 200 300 yl capacity pipet to remove beads from the 20x stock tube If necessary perform the volume transfer in 2 steps Do not use a 1 000 ul capacity pipet and or wide bore pipet tip 8 Protect the beads from light with aluminum foil Equilibrate to room temperature prior to use Preparing 1x coupled beads from 20x stock includes 20 excess volume Table 6 Premixed panel or one singleplex assay of Wells 20x Beads Wash Buffer pl Total Volume pl 96 288 5 472 5 760 48 144 2 736 2 880 Table 7 Mixing two singleplex assays 20x Beads ul 20x Beads ul Wash of Wells Singleplex 1 Singleplex 2 Buffer pl Total Volume pl 96 288 288 5 184 5 760 48 144 144 2 592 2 880 9 Cover unused wells of the assay plate with sealing tape 10 Prewet the filter plate Skip this step if using a flat bottom plate a Prewet the wells with 200 ul wash buffer and remove the liquid by vacuum filtration Dry the bottom of the filter plate thoroughly by blotting on a clean paper towel 11 Vortex the diluted 1x beads for 15 sec at medium speed Transfer 50 ul to each well of the assay plate 12 Wash the plate two times with 200 ul wash buffer according to your method of choice 17 13 Gat
12. crospheres and instrumentation licensed to Bio Rad Laboratories Inc by the Luminex Corporation POWERFUL CST antibodies developed and validated for ECCE Bio Plex cell signaling phosphoprotein and CONTENT target assays 34 Ordering Information Detailed ordering information can be found at www bio rad com bio plex Catalog Description Individual Components and Accessories Various Bio Plex Pro cell signaling singleplex sets 1 x 96 well 171 304006M Bio Plex Pro cell signaling reagent kit 1 x 96 well Various Bio Rad cell lysate controls pkg of 1 vial 171 304515 Bio Plex Pro cell signaling wash buffer for 1 x 96 well assay 330 ml 171 304502 Filter plate 1 x 96 well with clear plastic lid and tray Bio Plex x Plex Assays We Mix Create a premium custom assay using the online Bio Plex Assay Builder Go to www bio rad com bio plex assaybuilder to select analytes of interest Assays are supplied as premixed coupled beads and detection antibodies in the all in one kit format Bio Rad cell lysate controls are included 35 Life Science Group 10024929 Rev C Bio Rad Laboratories Inc Web site www bio rad com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699 Canada 905 364 3435 China 86 21 6169 8500 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 956965 Germany 089 31 8840 Greece 30 210 9532 220 Hong Kong 8
13. d SA PE should be prepared 10 min before use 19 27 28 29 Add the required volume of detection antibody diluent to an appropriately sized polypropylene tube This will be used to dilute SA PE to 1x Vortex the 100x stock of SA PE for 5 sec at medium speed Perform a quick spin to collect the entire volume at the bottom of the vial Dilute SA PE to 1x by pipetting the required volume into the tube containing detection antibody diluent Vortex and protect from light until ready to use Each well of the assay requires 0 5 ul of the 100x stock adjusted to a final volume of 50 ul in detection antibody diluent Table 10 Preparing 1x SA PE from 100x stock includes 25 excess volume of Wells 100x SA PE ul Detection Antibody Diluent pl Total Volume ul 96 60 5 940 6 000 48 30 2 970 3 000 30 31 32 33 34 35 After detection antibody incubation slowly remove and discard the sealing tape Wash the plate three times with 200 ul wash buffer according to your method of choice Vortex the diluted 1x SA PE at medium speed for 5 sec and transfer 50 pl to each well of the assay plate Cover with a new sheet of sealing tape and incubate in the dark for 10 min at room temperature with shaking Fully resuspend the beads SA PE mixture by shaking at 900 1 100 rpm for 30 sec Then turn down to 300 450 rpm for the specified incubation time After the streptavidin PE incubation step slowly rem
14. er HSP27 Ser S6 ribosomal protein EGF treated SK BR 3 171 YZ0003 Ser Ser 9 IGF 1R Tyr 8 IR B 46 IGF 1 treated HEK 293 171 YZ0005 IkB a Ser Ser Smad2 Ser Ser e ai E NF KB p65 Ser TNF a treated HeLa 171 YZ0008 p70 S6 Kinase Thr Ser p70 S6 Kinase Th NGF f treated PC12 171 YZ0006 BAD Ser 36 PDGFR a IRS 1 Ser 8 Ser 9 PDGFR p PDGF treated 171 YZ0007 mTOR Ser 9 PTEN Ser Stati Stats Tyr IFNo treated 171 YZ0004 Stat3 Ser a treated HeLa 000 Src Tyr Src transfected NIH8T3 171 20013 VEGFR 2 Tyr VEGF treated HUVEC 171 YZ0010 ZAP70 Tyr 9 H O treated Jurkat 171 YZ0012 Negative control for all phosphoprotein assays Phosphatase treated HeLa 171 YZBO01 11 continues Table 3 Selection guide for Bio Rad cell lysate controls continued Total Target Lysate Control Catalog Total Akt Total mTOR Total Erk 1 2 Total p38 MAPK Total GSK 3p Total p70 S6 Kinase E Total IkB a Total PTEN Untreated HeLa 171 YZTOO2 Total JNK Total Smad2 Total MEK1 Total IGF 1R Total Btk H O treated Ramos 171 YZ0011 Total c Jun Total CREB Untreated HEK 293 Total HER 2 EGF treated SK BR 3 171 YZ0003 Total Src Src transfected 171 YZ0013 Total ZAP 70 H O treated Jurkat 171 YZ0012 Negative control for all total target assays Detection antibody diluent House Keeping Pr
15. eted therapies with better efficacy and safety Bio Plex Pro cell signaling assays are magnetic bead based immunoassays for the detection of intracellular phosphoproteins and total target proteins in cell and tissue lysates The assays are available as singleplex sets which researchers can combine on their own to make a multiplex assay or as premixed all in one multiplex kits Phosphoprotein detection and total target detection are carried out in separate wells of a 96 well assay plate with just 1 10 ug of sample per well The assays have been optimized for exceptional sensitivity high specificity and improved performance over western blotting The use of magnetic MagPlex beads allows automation of wash steps on a Bio Plex Pro or similar wash station which greatly simplifies assay processing and improves assay precision For a complete list of all Bio Plex Pro cell signaling targets please visit www bio rad com bio plex References Chang L and Karin M 2001 Mammalian MAP kinase signalling cascades Nature 410 37 40 Principle Technology The Bio Plex suspension array system is built upon the three core elements of xMAP technology Fluorescently dyed microspheres also called beads each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension This allows simultaneous detection of up to 500 different types of molecules in a single well of the 96 well m
16. f clogging the instrument 4 If acquiring data from more than one plate empty the waste bottle and refill the sheath bottle after each plate if HTF is not present Select Wash Between Plates and follow the instructions Then repeat the Prepare Protocol and Acquire Data instructions 5 When data acquisition is complete select Shut Down and follow the instructions Reacquire Data It is possible to acquire data from a well or plate a second time using the Rerun Recovery mode located below Start in the Run Protocol step Any previous data will be overwritten unless the second run is saved under a different file name 1 Check the wells from which data will be reacquired 2 Aspirate the buffer with the wash method of choice but do not perform wash step 3 Add 100 ul of resuspension buffer to each well Cover the plate with a new sheet of sealing tape and shake plate at 900 1 100 rpm for 30 sec 4 Repeat the Acquire Data steps to reacquire data The data acquired should be similar to those acquired initially however the acquisition time will be extended because the wells have fewer beads 26 Luminex xPONENT Software Although guidelines are provided here consult the xPONENT software manual for more details Perform a system initialization with Luminex s calibration and performance verification kit as directed by Luminex Select Batches to set up the protocol and follow the information under Settings Note The instrument
17. from the Available list left and move to the Selected list right using the Add button To move all analytes at once simply click the Add All button If some of the analytes need to be removed from the Selected list highlight them and select Remove If desired it is possible to rename the panel by clicking Rename Panel and entering a new panel name Click Format Plate and format the plate according to the plate layout created in Section 2 Plan the Plate Layout To modify the plate layout follow the steps below see Figure 3 a b Select the Plate Formatting tab Select the blank icon and drag the cursor over all the wells that contain blanks Repeat this process for Controls e and Samples X Note that Bio Plex Manager automatically subtracts the blank MFI value from all other assay wells 23 Table 12 Bead regions for cell signaling assays Phosphoprotein Targets Bead Region Phosphoprotein Targets Bead Region Akt Ser 7 fis mTOR Ser 48 46 Akt Thre 75 NF KB p65 Ser 95 37 ATF 2 Thr 20 p38 MAPK Thr amp o Tyr amp 36 BAD Ser 5 26 p53 Ser 5 53 Btk Tyr 9 39 p70 S6 Kinase 89 55 C Abl Tyr 45 p70 S6 Kinase Thr Ser 55 c Jun Ser 56 p90 RSK Ser 35 CREB Ser 19 PDGFR a Tyr 28 EGFR Tyr 44 PDGFR B Br EGFR Tyr 3 44 PISK p85 Tyr 5 54 E EAE Theyre 3s PTEN Ser 22 GSK 3o p Ser Ser 18 S6 ribosomal protein
18. he wells to prevent leaking from the bottom 15 4 Run the Assay Considerations a Bring all assay components and samples to room temperature before use a Use calibrated pipets and pipet carefully avoiding bubbles Assay incubations are carried out in the dark Cover the plate with aluminum foil or otherwise protect from extended exposure to light DAY 1 Prepare Samples and Controls 1 Thaw sample lysates and keep on ice see Section 1 Prepare the Samples for lysate preparation Reconstitute lyophilized cell lysate control with 250 ul of diH O vortex for 5 sec to mix and incubate at room temperature for 20 min Protein concentration is now 200 ug ml Unused lysate can be stored at 20 C for 3 months Centrifuge all samples and lysate controls at 15 000 x g for 10 min at 4 C before dispensing to wells Prepare and Add Coupled Beads and Samples 4 Use Tables 6 and 7 or the Calculation Worksheet on pages 32 33 as a reference to calculate the volume of coupled beads and wash buffer needed Add the required volume of wash buffer to an appropriately sized polypropylene tube This will be used to dilute beads to 1x Vortex the 20x stock of coupled beads at mid speed for 30 sec Carefully open the cap and pipet any liquid trapped in the cap back into the tube This is important to ensure maximum bead recovery Do not centrifuge the vial doing so will cause the beads to pellet 16 7 Dilute coupled beads to 1x
19. her the samples Bio Rad cell lysate controls and blank Use detection antibody diluent as the blank Transfer 50 ul of each sample or blank to the appropriate wells of the plate 14 Cover with a new sheet of sealing tape and incubate in the dark overnight 15 18 hr at room temperature with shaking Note Fully resuspend the beads sample mixture by vigorously shaking at 900 1 100 rpm for 30 sec Slowly ramp up to speed to avoid splashing Then turn down to 300 450 rpm for the specified incubation time DAY 2 Prepare Instrument and Wash Method 15 Start up warm up and calibrate the Bio Plex system as described in Section 5 Read the Plate This may take up to 30 min 16 Meanwhile bring all buffers and diluents to room temperature 17 Prepare the wash method as described in Section 3 Prepare the Wash Prepare and Add Detection Antibodies 18 Use Tables 8 and 9 or the Calculation Worksheet on pages 32 33 to calculate the volume of detection antibodies and Bio Plex detection antibody diluent needed Detection antibodies should be prepared 10 min before use 19 Add the required volume of Bio Plex detection antibody diluent to an appropriately sized polypropylene tube 20 Vortex the 20x stock of detection antibodies for 15 20 sec at medium speed then perform a quick spin to collect the entire volume at the bottom of the tube 21 Dilute detection antibodies to 1x by pipetting the required volume into the tube containi
20. ial at different angles for 30 sec at medium speed before aliquoting beads Adjust pressure to 1 to 3 Hg Generally 100 ul liquid should take 3 4 sec to clear from the well Adjust the needle height to coincide with the plate type provided in the kit Use recommended magnetic washer settings with correct soaking time 29 continues Table 13 Troubleshooting guide continued Problem Possible Causes Possible Solutions High background Prolonged incubation of Follow the procedure incubation detection antibodies and or ime precisely streptavidin PE Wash steps performed Perform washes as described in incorrectly or insufficient he assay instructions washing volume High assay CV Bottom of filter plate not dry Dry the bottom of the filter plate Contamination with wash buffer during wash steps Cell debris in lysate not cleared Shaking speed too high during assay incubation Reagents and assay components not equilibrated to room temperature with absorbent paper towel preferably lint free to prevent cross well contamination Be careful not to splash wash buffer from well to well Filter wells completely to remove residual buffer if using filter plate Reduce microplate shaking speed to minimize splashing Centrifuge at 15 000 x g for 10 min at 4 C to remove cellular debris Fully resuspend bead mixture at 900 1 100 rpm for 30 sec then turn down to 300 450 rpm for the specified incubation time B
21. icroplate on the Bio Plex 3D system up to 100 different types of molecules on the Bio Plex 200 system and up to 50 different types of molecules on the Bio Plex MAGPIX system A dedicated plate reader The Bio Plex 200 and Bio Plex 3D systems are flow cytometry based instruments with two lasers and associated optics to measure the different molecules bound to the surface of the beads In the Bio Plex MAGPIX system the entire sample load volume is injected into a chamber where the beads are imaged using LED and CCD technology A high speed digital signal processor that efficiently manages the fluorescence data Assay Format Bio Plex Pro assays are essentially immunoassays formatted on magnetic beads The assay principle is similar to that of a sandwich ELISA Figure 1 Capture antibodies directed against the desired biomarker are covalently coupled to the beads Coupled beads react with the sample containing the biomarker of interest After a series of washes to remove unbound protein a biotinylated detection antibody is added to create a sandwich complex The final detection complex is formed with the addition of streptavidin phycoerythrin SA PE conjugate which serves as a fluorescent indicator or reporter Biomarker of V Streptavidin Q o lus np Fluorescent Capture Biotinylated Reporter Antibody Detection Antibody Fig 1 Bio Plex sandwich immunoassay Data Acquisition and Analys
22. imental samples antibody or streptavidin PE and Bio Rad cell lysate controls Expired Bio Plex reagents were used Incorrect incubation temperature Insufficient incubation time Beads lost Verify sample protein concentration is within assay range Optimization of protein concentration may be needed based on targeted protein expression level Prepare fresh lysate accordingly Check the calculations and be careful to add the correct volumes Check that reagents have not expired Use new or non expired components Incubations should be at room temperature 20 22 C Adhere to the recommended incubation time Use recommended magnetic washer settings with correct magnet soaking time 28 continues Table 13 Troubleshooting guide continued Problem Possible Causes Possible Solutions Low bead count Cell debris in lysate not cleared Assay plate not shaken enough prior to reading Clogged reader Miscalculation of bead dilution Clumping of stock beads in vials Vacuum setting too high during suction of assay plate Reader needle height incorrect Beads lost Centrifuge at 15 000 x g for 10 min at 4 C to remove cellular debris Shake plate at 900 1 100 rpm for 30 sec before data acquisition Refer to the troubleshooting guide in the Bio Plex system hardware instruction manual bulletin 10005042 Check the calculations and be careful to add the correct volumes Vortex the stock v
23. ing approximately 20 strokes Note It may be necessary to lyse the samples with different volumes of cell lysis solution to obtain the specified protein concentration range 3 Transfer the ground tissue to a clean microcentrifuge tube and freeze sample at 70 C Freezing and thawing samples helps increase cell lysis effects 4 Thaw the sample and sonicate on ice for example with a Sonifier 450 Duty Cycle 40 Output 1 Pulse Sonicating 18x 5 Remove insoluble cellular matter as in Adherent Cells step 5 above 6 Follow Adherent Cells steps 6 9 above 10 Bio Rad Cell Lysate Controls The positive and negative cell lysate controls are used for qualitative verification of assay performance Refer to Table 3 to select the appropriate controls for your phosphoprotein or total target of interest Table 3 Selection guide for Bio Rad cell lysate controls Phosphoprotein of Interest Lysate Control Catalog Akt Ser GSK 3o p Ser Ser Akt Thr 99 MEK1 Ser 7 Ser EGF treated HEK 293 171 YZ0001 Erk1 2 Thr Tyr Thri amp Tyr 8 ATF 2 Thr JNK Thr 8 Tyr 85 c Jun Ser p38 MAPK Thr Tyr amp UV treated HEK 293 171 YZ0009 CREB 33 p53 Tyr PISK p85 H O treated R 171 YZ0011 Lyn Syk Tyr O treated Ramos 00 c Abl Tyr 5 Untreated K 562 171 2 003 EGFR EGFR Tyr 3 EGF treated HeLa 171 YZ0002 HER 2 Tyr p90 S
24. is Data from the reactions are acquired using a Bio Plex system or similar Luminex based reader When a multiplex assay suspension is drawn into the Bio Plex 200 reader for example a red 635 nm laser illuminates the fluorescent dyes within each bead to provide bead classification and thus assay identification At the same time a green 532 nm laser excites PE to generate a reporter signal which is detected by a photomultiplier tube PMT A high speed digital processor manages data output and Bio Plex Manager software presents data as median fluorescence intensity MFI The relative concentration of analyte bound to each bead is proportional to the MFI of the reporter signal Using Bio Plex Data Pro software data from multiple instrument runs can be combined into a single project for easy data management quick visualization of results and simple statistical analysis Kit Components and Storage Table 1 Components required for a complete 1 x 96 well cell signaling assay Note that custom x Plex assays include a premixed multiplex set of beads and detection antibodies in an all in one kit kkk Component Quantity Singleplex or Multiplex Set Coupled magnetic beads 20x 1 tube Detection antibodies 20x 1 tube Cell Signaling Reagent Kit catalog 171 304006M Cell wash buffer 1 bottle 50 ml Cell lysis buffer 1 bottle 50 ml Cell lysis factor QG 1 vial Wash buffer 1 bottle 330 ml Detection antibody diluent 1
25. ng detection antibody diluent Vortex Each well of the assay requires 1 25 ul of the 20x stock adjusted to a final volume of 25 ul in detection antibody diluent 18 Preparing 1x detection antibodies from 20x stock includes 25 excess volume Table 8 Premixed panel or one singleplex assay 20x Detection Detection Antibody of Wells Antibodies pl Diluent pl Total Volume pl 96 150 2 850 3 000 48 75 1 425 1 500 Table 9 Mixing two singleplex assays 20x Detection 20x Detection Detection Antibodies Antibodies pl Antibody of Wells Singleplex 1 Singleplex 2 Diluent pl Total Volume pl 96 150 150 2 700 3 000 48 75 75 1 350 1 500 22 After the overnight incubation slowly remove discard the sealing tape 23 Wash the plate three times with 200 ul wash buffer according to your method of choice 24 Vortex the diluted 1x detection antibodies gently for 5 sec and transfer 25 ul to each well of the assay plate 25 Cover with a new sheet of sealing tape and incubate in the dark for 30 min at room temperature with shaking Fully resuspend the beads detection antibody mixture by shaking at 900 1 100 rpm for 30 sec Then turn down to 300 450 rpm for the specified incubation time Prepare and Add Streptavidin PE SA PE 26 While detection antibodies are incubating use Table 10 or the Calculation Worksheet on pages 32 33 to calculate the volume of SA PE and detection antibody diluent neede
26. otein Human GAPDH p Actin Negative control for all total target assays Untreated HeLa 171 YZT002 Detection antibody diluent 2 Plan the Plate Layout Prior to running the assay determine the total number of wells in the experiment using the Plate Layout Template on page 30 or the Plate Formatting tab in Bio Plex Manager A suggested plate layout is shown in Figure 2 with all conditions in duplicate Please note that the Bio Plex 200 instrument reads the plate vertically 12 1 Assign blank negative control positive control and samples accordingly Note When designating blank using lt gt Bio Plex Manager software will automatically subtract the blank MFI value from all other assay wells 2 Once the total number of wells is known see Tables 6 9 or the Calculation Worksheet on pages 32 33 to determine the required volumes of beads detection antibodies and streptavidin PE to use Note that 20 25 excess volume is included in the calculations to compensate for transfer loss Legend J Bio Ples ager t Prot 2 File Edit View Format Options Window Help ose 5 Ps Blank Protocol Settings Plate Formatting Plate Groupings x Samples Controls s ep Z nnumn EI EI ag A FEE RIE 9 E spe s s s s e sea s s EEk BEBE e ET Fig 2 S
27. ove and discard the sealing tape Wash the plate three times with 200 ul wash buffer according to your method of choice 20 36 To resuspend beads for plate reading add 125 ul resuspension buffer to each well Cover the plate with a new sheet of sealing tape and shake at 900 1 100 rpm for 30 sec 36 Slowly remove the sealing tape and place the plate on the reader to acquire data Table 11 Read the plate using the appropriate instrument settings Instrument RP1 PMT DD Gates Bead Events Bio Plex 100 200 High 5 000 low 25 000 high 50 Bio Plex 3D Enhanced Select MagPlex beads 50 Bio Plex MAGPIX N A use default instrument settings Or similar Luminex based system 5 Read the Plate Bio Plex Manager software is recommended for all Bio Plex Pro assay data acquisition and analysis Instructions for Luminex xPONENT software are also included For instructions on using other x MAP system software packages contact Bio Rad Technical Support or your regional Bio Rad field applications specialist Prepare Instrument Start up and calibrate the Bio Plex 100 200 or similar system with Bio Plex Manager software prior to setting up the assay The calibration kit should be run daily or before each use of the instrument to standardize the fluorescent signal To prepare either a Bio Plex 3D or Bio Plex MAGPIX reader consult its respective user manual For instructions on using other xMAP system software packages
28. p and calibration can be performed together by selecting the Start up and calibrate icon 6 Prepare Protocol in Bio Plex Manager Software Version 6 0 and Higher The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete A protocol file specifies the analytes in the assay the plate wells to be read sample information and instrument settings Bio Plex Manager software versions 6 0 and higher contain protocols for most Bio Plex assays Choose from available protocols or create a new 22 protocol To create a new protocol select File then New from the main menu Locate and follow the steps under Protocol Settings 6 Click Describe Protocol and enter information about the assay optional Click Select Analytes and create a new panel Visually confirm the selected analytes and proceed to step 8 a Click the Add Panel button 4 in the Select Analytes toolbar Enter a new panel name Select Bio Plex Pro Assay Magnetic or MagPlex Beads Magnetic from the assay dropdown list If using Bio Plex Manager version 5 0 or lower select MagPlex from the assay dropdown list Click the Add button Enter the bead region number and name for the first analyte Click Add Continue to repeat for each analyte in the assay Refer to the bead regions listed in Table 12 Click the Add button when the last analyte has been added and click OK to save the new panel Highlight analytes
29. pos o o s n i 1 12 120129 o n n CIE OOOee DBBBBBDD ES D E F G H Fig 3 Plate formatting 10 Click Enter Sample Info and enter sample information and the appropriate dilution factor if any 11 Click Run Protocol and confirm that the assay settings are correct a Refer to Table 11 for the recommended RP1 PMT setting Protocols using alternative settings should be validated by the end user b Confirm that data acquisition is set to 50 beads per region In Advanced Settings confirm that the bead map is set to 100 region the sample size is set to 50 pl and the doublet discriminator DD gates are set to 5 000 Low and 25 000 High In Bio Plex Manager software versions 4 0 4 1 4 1 1 and 5 0 check Override Gates and set the DD gate values as indicated c Select Start name and save the rbx file and begin data acquisition The Run Protocol pop up screen will appear Click Eject Retract to eject the plate carrier 25 Acquire Data 1 Shake the assay plate at 900 1 100 rpm for 30 sec and visually inspect the plate to ensure that the assay wells are filled with buffer Slowly remove the sealing tape before placing the plate on the plate carrier 2 Click Run Protocol and on the pop up screen select Load Plate and click OK to start acquiring data 3 Use the Wash Between Plates command after every plate run to reduce the possibility o
30. quivalent capable of 300 1 100 rpm Aurum vacuum manifold For vacuum filtration BR 2000 vortexer Reagent reservoirs 25 ml For capture beads and detection antibodies Reagent reservoir 50 ml for reagents and buffers Pall Life Science Acrodisc 32 mm PF syringe filter 0 45 um Supor membrane DC protein assay kit II Phenylmethylsulfonyl fluoride PMSF Dimethyl sulfoxide DMSO Kontes tissue grinder Bulletin 10024930 download at www bio rad com bio plex Bio Rad catalog 1t17 1 000205 Bio Rad catalog 117 1 203001 Bio Rad catalog 1t17 1 203060 Bio Rad catalog 300 34376 Bio Rad catalog 1t17 1 020100 Bio Rad catalog 117 1 025001 IKA catalog 320 8000 VWR catalog 57019 600 Bio Rad catalog 732 6470 Bio Rad catalog 166 0610 VistaLab catalog 3054 1002 2 catalog 3054 1004 VistaLab catalog 3054 1006 Pall Life Sciences catalog 4654 Bio Rad catalog 500 0112 Sigma catalog P7626 Sigma catalog D2650 VR catalog KT885000 0002 Other Polypropylene tubes for reagent dilutions calibrated pipets pipet tips sterile distilled water aluminum foil paper towels 1 5 or 2 ml microcentrifuge tubes and a standard flat bottom microplate for calibrating vacuum manifold Assay Workflow Prewet wells for filter plate only Add 50 yl 1x beads to wells Wash 2x Add 50 ul samples controls and blanks incubate overnight at RT with shaking Add 25 ul 1x detection antibod
31. ring all reagents and assay components to room temperature prior to dispensing 30 Plate Layout Template ck LL OL m lt 31 Calculation Worksheet If using either a premixed panel or one singleplex assay follow these instructions Plan the plate layout and enter the number of wells to be used in the assay 1 Determine the volume of 1x coupled beads needed a Each well requires 50 ul of coupled beads 1x x 50 ul ul b Include 20 excess to ensure enough volume m ul x 0 20 ul c Total volume of 1x coupled beads ul ul i ul d Volume of 20x coupled beads stock ul 20 ul e Volume of wash buffer required z z ul _ ul 2 Determine the volume of 1x detection antibody needed a Each well requires 25 ul detection antibodies 1x x25yl ul b Include 25 excess to ensure enough volume ul x 0 25 ul c Total volume of 1x detection antibodies ul ul ul d Volume of 20x detection antibodies ul 20 ul e Volume of detection antibody diluent required z ul ul ul 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 yl streptavidin PE 1 x 50 ul ul 1 12 b Include 25 excess to ensure enough volume ul x 0 25 ul 12 13 c Total volume of 100x streptavidin PE ul ul ul 12 13 14 d Volume of 100x streptavidin PE required ul 100 ul 14 15 e Volume of detec
32. the lysate at 15 000 x g for 10 min at 4 C using a benchtop microcentrifuge 6 Collect the filtered lysate or supernatant after centrifugation 7 Measure protein concentration using Bio Rad s DC protein assay kit and if needed adjust protein concentration with cell lysis buffer containing PMSF and cell lysis factor QG 8 The suggested working protein concentration range for Bio Plex cell signaling assays is 3 200 pg ml 0 15 10 ug per assay well 9 Store the aliquoted lysates at 70 C until ready to use Suspension Cells 1 Collect cell suspension and pellet the cells by spinning at 1 000 x g for 5 min at 4 C 2 Aspirate off cell culture medium completely 3 Wash by resuspending the cells with ice cold cell wash buffer bottle with the blue cap 4 Centrifuge the cells at 1 000 x g for 5 min at 4 C 5 Completely remove the buffer 6 Immediately add the proper volume of cell lysis buffer and gently rock for 20 min at 4 C 7 Remove insoluble cellular particulates as described in Adherent Cells step 5 above 8 Follow Adherent Cells steps 6 9 above Tissue Samples 1 Cut the tissue into small pieces 3 x 3 mm for ease of handling and blood removal If necessary wash the tissue with ice cold cell wash buffer bottle with the blue cap to completely remove all blood Then transfer the tissue to a 2 ml tissue grinder 2 Add an adequate volume of cell lysis solution and grind the tissue sample on ice us
33. tion antibody diluent required ul ul 14 15 16 32 If mixing two or more singleplex assays follow these instructions Enter the number of wells to be used in the assay 1 1 Determine the volume of 1x coupled beads needed a Each well requires 50 ul coupled beads 1x x 50 ul ul 1 2 b Include 20 excess to ensure enough volume ul x 0 20 ul 2 3 c Total volume of 1x coupled beads ul ple ul 2 3 4 d Enter the number of singleplex sets or analytes that will be multiplexed 5 e Volume of 20x coupled beads required from each stock tube ul 20 ul 6 f Total volume of bead stock required x ul ul 5 6 7 g Volume of wash buffer required ul ul ul 4 7 8 2 Determine the volume of 1x detection antibody needed Each well requires 25 ul detection antibodies 1x x 25 ul 1 9 b Include 25 excess to ensure enough volume ul x 0 25 ul 9 10 c Total volume of 1x detection antibodies ul ulis ul 9 10 11 d Enter the number of singleplex sets or analytes that will be multiplexed 5 e Volume of 20x detection antibodies required from each stock tube ul 20 ul 12 f Total volume of combined detection antibody stock ul x ul 12 5 13 g Volume of detection antibody diluent required ul ul ul 11 13 14 3 Determine the volume of 1x streptavidin PE needed a Each well requires 50 ul streptavidin PE 1 x 50 ul ul 1 15 b Include
34. uggested plate layout For detailed instructions on plate formatting in Bio Plex Manager see Section 5 Read the Plate 13 3 Prepare the Wash Method The cell signaling assays are compatible with both magnetic separation and vacuum filtration methods However for best results we recommend performing the assays in a flat bottom plate with magnetic separation Table 4 Summary of compatible wash stations and plate types Wash Method Wash Station Assay Plate Magnetic separation Bio Plex Pro Flat bottom plate Bio Plex handheld magnetic washer Vacuum filtration Vacuum manifold manual Filter plate Setting up the Bio Plex Handheld Magnetic Washer Place an empty flat bottom plate on the magnetic washer by sliding it under the retaining clips Push the clips inward to secure the plate Make sure the plate is held securely For detailed instructions refer to the user guide bulletin 10023087 Setting up the Bio Plex Pro The wash station should be primed before use See bulletin 5826 1 Install the appropriate plate carrier on the wash station 2 Use the Prime procedure to prime channel 1 with wash buffer Note Before using the Bio Plex Pro wash station make sure to define edit a program with the correct settings for cell signaling assays Table 5 a Existing cytokine assay programs MAGx2 MAGx8 VACx2 and VACx3 should not be used Refer to the wash station instruction manual bulletin 10013125 or contact Bio
35. y incubate 30 min at RT with shaking Wash Add 50 ul 1x streptavidin PE incubate 10 min at RT with shaking Wash 3x Add 125 ul of resuspension buffer shake for 30 sec Acquire data Important Considerations Assay Procedures a Please pay close attention to vortexing shaking and incubation times and to Bio Plex reader PMT RP1 setting as these have been optimized specifically for the cell signaling assays For optimal performance use only reagents specific for Bio Plex Pro cell signaling assays Reagents in other Bio Plex assay panels have not been validated for use in the cell signaling assays a Do not reuse diluted 1x coupled beads detection antibodies or streptavidin PE a Wash as outlined in Table 5 Incomplete washes may cause assay variation a f the data are not acquired immediately the assay plate may be stored at 4 C for up to 24 hr protected from light Assay Quick Guide Each assay kit comes complete with a printed Bio Plex Pro Assay Quick Guide bulletin 10024930 which can be used to prepare and run a full 1 x 96 well assay plate Users can also download a copy at www bio rad com bio plex Bead Regions and Multiplexing Compatibility Bead regions for all analytes are listed in Table 12 a Compatible singleplex assays may be mixed to create a multiplex assay a Do not mix phosphoprotein assays with corresponding total target assays for example phospho Akt and total Akt Instruments and
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