Human Angiogenesis Base Kit A
Contents
1. Also use separate reservoirs for each reagent e To ensure accurate results proper adhesion of plate sealers during incubation steps is necessary e Protect microparticles and Streptavidin PE from light at all times to prevent photobleaching 2 For research use only Not for use in diagnostic procedures MATERIALS PROVIDED amp STORAGE CONDITIONS Store the unopened kit at 2 8 C Do not use past kit expiration date STORAGE OF OPENED DILUTED PART PART DESCRIPTION OR RECONSTITUTED MATERIAL Standard Cocktail 893605 2 vials of recombinant human protein biomarkers in a buffered protein base with preservatives lyophilized Microparticle 895575 6mL ofa buffered protein base with May be stored for up to 1 month at 2 8 C Diluent 5 preservatives Once diluted any unused microparticle cocktail must be discarded Biotin Antibody 895832 5 5 mLofa buffered protein base with Diluent 2 preservative Calibrator Diluent 895580 2vials 21 mL vial of a buffered protein base RD6 49 with preservatives Used undiluted for serum plasma human milk samples Used diluted 2 1 for cell culture supernate urine samples Must be warmed to room temperature prior to use May be stored for up to 1 month at 2 8 C Streptavidin PE 892525 0 07 mL ofa 100 fold concentrated streptavidin phycoerythrin conjugate with preservatives Wash Buffer 895003 21 mL ofa25 fold concentrated solution of Concentrate buffered surfactant with preserva2. 751 All trademarks and registered trademarks are the property of their respective owners www RnDSystems com PLATE LAYOUT ut to record standards and samples assayed ECX X XXXXX ECX XX XXXX OOOO 00 NOOOOOOO 6 OOOOUOOOO NOOO OO0O0T QOOOOOOO OOOOOOOO NOOO OO0O0T 6 OOOOOOOO OOOOOUOOOO 7
3. 0 uL of sample 200 uL of Calibrator Diluent RD6 49 Note Angiogenin milk samples require an additional 4 fold dilution A suggested 4 fold dilution is 100 uL of diluted sample 300 uL of Calibrator Diluent RD6 49 to complete the 20 fold dilution Mix thoroughly REAGENT PREPARATION Bring all reagents to room temperature before use Wash Buffer If crystals have formed in the concentrate warm to room temperature and mix gently until the crystals have completely dissolved Add 20 mL of Wash Buffer Concentrate to deionized or distilled water to prepare 500 mL of Wash Buffer Calibrator Diluent RD6 49 diluted 2 1 For cell culture supernate and urine samples Add 20 mL of Calibrator Diluent RD6 49 to 10 mL of deionized or distilled water to prepare 30 mL of Calibrator Diluent RD6 49 diluted 2 1 Standard Reconstitute the Standard Cocktail with Calibrator Diluent RD6 49 for serum plasma and human milk samples or Calibrator Diluent RD6 49 diluted 2 1 for cell culture supernate and urine samples Refer to the Standard Value Card for the reconstitution volume and assigned values Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions Use reconstituted standards within 4 hours Use polypropylene tubes Pipette 500 uL of the reconstituted Standard into a tube labeled Standard 1 Pipette 300 uL of the appropriate Calibrator Diluent into the remaining tubes Use Standard 1 to produce a
4. 4 fold dilution series below Refer to analyte specific datasheets for details Mix each tube thoroughly before the next transfer Standard 1 serves as the high standard The appropriate Calibrator Diluent serves as the blank 100 uL 100uL 100 uL 100uL 100 pL 100 uL PA A OOO 500 ul Std p p p p D Standard Cocktail Standard 1 Standard2 Standard3 Standard4 Standard5 Standard6 Standard 7 www RnDSystems com 5 DILUTED MICROPARTICLE COCKTAIL PREPARATION 1 Centrifuge each Microparticle Concentrate vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vials to resuspend the microparticles taking precautions not to invert the vials 3 Dilute the Microparticle Concentrates in the mixing bottle provided The volume of the Microparticle Concentrate listed in the table below is for each analyte e g if measuring a full plate of FGF basic and VEGF add 50 uL of FGF basic Microparticle Concentrate and 50 uL of VEGF Microparticle Concentrate to 5 mL of Microparticle Diluent Number of Wells Used Microparticle Concentrate Microparticle Diluent 96 50 0 uL 5 00 mL 72 37 5 uL 3 75 mL 48 25 0 uL 2 50 mL 24 12 5 uL 1 25 mL Note Protect microparticles from light during handling Diluted microparticles cannot be stored Prepare microparticles within 30 minutes of use DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION 1 Centrifuge each Biotin Antibody Concentrate vial for 30 seconds at 1000 x g pr
5. LT oigear n senses E EER EEN E O ESE CAEBRACION orrie a E E E E E S REFERENCE oerrinne E ENE EAA AA A A E PEATE EAT OU Toae A EAA A E E MANUFACTURED AND DISTRIBUTED BY USA amp Canada R amp D Systems Inc 614 McKinley Place NE Minneapolis MN 55413 USA TEL 800 343 7475 612 379 2956 FAX 612 656 4400 E MAIL info RnDSystems com DISTRIBUTED BY UK amp Europe R amp D Systems Europe Ltd 19 Barton Lane Abingdon Science Park Abingdon OX14 3NB UK TEL 44 0 1235 529449 FAX 44 0 1235 533420 E MAIL info RnDSystems co uk China R amp D Systems China Co Ltd 24A1 Hua Min Empire Plaza 726 West Yan An Road Shanghai PRC 200050 TEL 86 21 52380373 FAX 86 21 52371001 E MAIL info RnDSystemsChina com cn INTRODUCTION Angiogenesis involving the sprouting and branching of new blood vessels from pre existing vessels plays a critical role in wound healing and tumor growth The typically quiescent adult vasculature does not require ongoing angiogenesis except in female reproductive organs or in response to injured tissue Pathologic angiogenesis occurs in tumor development since the generation of a tumor blood supply is a rate limiting step in tumor progression and metastasis and in other vasculature disorders 1 In addition angiogenesis also represents an excellent therapeutic target for the treatment of cardiovascular disease 2 The emergence and maturation of new vessels are complex and highly regul
6. Magnetic Luminex Performance Assay Human Angiogenesis Base Kit A Catalog Number LANM000 For the simultaneous quantitative determination of multiple human protein biomarker concentrations in cell culture supernates serum platelet poor plasma urine and human milk This package insert must be read in its entirety before using this product For research use only Not for use in diagnostic procedures TABLE OF CONTENTS SECTION INTRODUC TION reren a E E caeves eecersseusseeecstevtaeaceaese PRINCIP EE fe THEA SA V peenar A A A AAA LUMITATONS OF THE PROCEDURE siseserescrntireirein aena ee n E N NE TE CRNCA E EN ea a E EE E E N E MATERIALS PROVIDED amp STORAGE CONDITIONS sssesssssesssessesssessssssesseessessseoseessesseesseeseess OTHER JUR PEIE SRE OURE D eenean e E E EE EEE E EE PRECAUTION Serrar EEE E E N SAMPLE COLLECTION AND STORAGE ssecaseasrerasscsennaewasnsremmiiaiacteaenmnnanetnsoimnienen SAMPEEPREPA RATION saree EA EEE eee estaranusienacs REA GENTPRERARA TION an ea A E E E ORTE DILUTED MICROPARTICLE COCKTAIL PREPARATION s ssesssesssessesssesseeseesseessesseessesssesseesss DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION ssssssssesssessesssesseessesseessesseessesssessessss STREPTAVIDIN PE PREPARATION ssssssssesesessesssessseseessseoseosseoseesscoseesseesseossesseossesseosscsseosscosecsseessees INSTRUMENT SETTING Sirenin AEn A E EEE EEAO tenets terse PSI PROCEDUR E correr prana T E E E E EE E ersten CA CULANON OF RESU
7. ated processes that require multiple signaling cascades and affect endothelial cell proliferation and migration 3 Activators and inhibitors of angiogenesis coordinate the angiogenic balance which dictates whether an endothelial cell will be quiescent or angiogenic Positive regulators of angiogenesis include FGFs VEGFs PDGF BB and EGF Negative regulators include thrombospondin 1 angiostatin and endostatin Factors such as VEGF placenta growth factor PIGF and Angiopoietin 1 stimulate angiogenesis as well as the de novo incorporation of marrow derived endothelial cells into the walls of growing vessels 1 3 When combined with separately available analyte specific bead sets this kit is an excellent tool for simultaneously assessing the levels of multiple pro and anti angiogenic molecules in a single sample Any combination of the following bead sets are suitable for use with this base kit Analyte Catalog Number Microparticle Region Angiogenin LANM265 12 Angiopoietin 1 LANM923 25 Endostatin LANM1098 14 FGF acidic LANM232 15 FGF basic LUHM233 13 PIGF LANM264 20 PDGF AA LANM221 18 PDGF BB LANM220 19 Thrombospondin 2 LANM1635 21 VEGF LUHM293 39 VEGF D LANM622 22 www RnDSystems com 1 PRINCIPLE OF THE ASSAY Magnetic Luminex Performance Assay multiplex kits are designed for use with the Luminex MAGPIX CCD Imager Alternatively kits can be used with the Luminex 100 200 or Bio Rad Bio Plex dual laser flow based s
8. c range of the assay further dilute the samples with the appropriate Calibrator Diluent and repeat the assay e Any variation in standard diluent operator pipetting technique washing technique incubation time or temperature and kit age can cause variation in binding e Variations in sample collection processing and storage may cause sample value differences e This assay is designed to eliminate interference by other factors present in biological samples Until these factors have been tested in the Luminex Performance Assay the possibility of interference cannot be excluded e Magnetic Luminex Performance Assays afford the user the benefit of multianalyte analysis of biomarkers in a single complex sample For each sample type a single multipurpose diluent is used to optimize recovery linearity and reproducibility Such a multipurpose diluent may not optimize any single analyte to the same degree that a unique diluent selected for analysis of that analyte can optimize conditions Therefore some performance characteristics may be more variable than those for assays designed specifically for single analyte analysis e Only the analytes listed on the Standard Value Card can be measured with this base kit TECHNICAL HINTS e When mixing or reconstituting protein solutions always avoid foaming e To avoid cross contamination change pipette tips between additions of each standard level between sample additions and between reagent additions
9. id breathing mist Wear protective gloves clothing eye and face protection Wash hands thoroughly after handling Please refer to the MSDS on our website prior to use SAMPLE COLLECTION AND STORAGE The sample collection and storage conditions listed below are intended as general guidelines Sample stability has not been evaluated Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Serum Use a serum separator tube SST and allow samples to clot for 30 minutes at room temperature before centrifuging for 15 minutes at 1000 x g Remove serum and assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Platelet poor Plasma Collect plasma on ice using heparin or EDTA as an anticoagulant Centrifuge for 15 minutes at 2 8 C at 1000 x g within 30 minutes of collection An additional centrifugation step of the separated plasma at 10 000 x g for 10 minutes at 2 8 C is recommended for complete platelet removal Assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles Note Citrate plasma has not been validated for use in this assay Angiopoietin 1 PDGF AA and PDGF BB are present in platelet granules and are released upon platelet activation Therefore to measure circulating levels of these factors platelet free plasma should be collected fo
10. in step 4 Add 50 uL of diluted Streptavidin PE to each well Securely cover with a foil plate sealer and incubate for 30 minutes at room temperature on the shaker set at 800 50 rom Repeat the wash as in step 4 Resuspend the microparticles by adding 100 uL of Wash Buffer to each well Incubate for 2 minutes on the shaker set at 800 50 rpm Read within 90 minutes using a Luminex or Bio Rad analyzer Note Resuspend microparticles immediately prior to reading Samples require dilution See the Sample Preparation section For research use only Not for use in diagnostic procedures CALCULATION OF RESULTS Use the Standard concentrations on the Standard Value Card and calculate 4 fold dilutions for the remaining levels Average the duplicate readings for each standard and sample and subtract the average blank Median Fluorescence Intensity MFI Create a standard curve for each analyte by reducing the data using computer software capable of generating a five parameter logistic 5 PL curve fit Since samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor CALIBRATION This assay is calibrated against highly purified recombinant human protein biomarkers produced at R amp D Systems REFERENCES 1 Bergers G and L E Benjamin 2003 Nat Rev Cancer 3 401 2 Atluri P and Y J Woo 2008 BioDrugs 22 209 3 Karamysheva A F 2008 Biochemistry Mosc 73
11. ior to removing the cap 2 Gently vortex the vials taking precautions not to invert the vials 3 Add 50 uL of each Biotin Antibody Concentrate to the vial of Biotin Antibody Diluent 2 Mix gently STREPTAVIDIN PE PREPARATION Use a polypropylene amber bottle or a polypropylene tube wrapped with aluminum foil Protect Streptavidin PE from light during handling and storage 1 Centrifuge the Streptavidin PE vial for 30 seconds at 1000 x g prior to removing the cap 2 Gently vortex the vial taking precautions not to invert the vial 3 Dilute the 100X Streptavidin PE to a 1X concentration by adding 55 uL of Streptavidin PE to 5 5 mL of Wash Buffer 6 For research use only Not for use in diagnostic procedures INSTRUMENT SETTINGS Luminex MAGPIX analyzer a Assign the microparticle region for each analyte being measured see Introduction on page 1 b 50 events bead c Sample size 50 uL d Collect Median Fluorescence Intensity MFI Luminex 100 200 and Bio Rad Bio Plex analyzers Note Calibrate the analyzer using the proper reagents for superparamagnetic microparticles refer to instrument manual a Assign the bead region for each analyte being measured see Introduction on page 1 b 50 events bead c Minimum events 0 d Flow rate 60 uL minute fast e Sample size 50 uL f Doublet Discriminator gates at approximately 8000 and 16 500 g Collect MFI Note The CAL2 setting for the Bio Rad Bio Plex analyze
12. orting and detection platforms Analyte specific antibodies are pre coated onto color coded magnetic microparticles Microparticles standards and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest After washing away any unbound substances a biotinylated antibody cocktail specific to the analytes of interest is added to each well Following a wash to remove any unbound biotinylated antibody streptavidin phycoerythrin conjugate Streptavidin PE which binds to the biotinylated antibody is added to each well A final wash removes unbound Streptavidin PE the microparticles are resuspended in buffer and read using the Luminex MAGPIX Analyzer A magnet in the analyzer captures and holds the superparamagnetic microparticles in a monolayer Two spectrally distinct Light Emitting Diodes LEDs illuminate the microparticles One LED identifies the analyte that is being detected and the second LED determines the magnitude of the PE derived signal which is in direct proportion to the amount of analyte bound Each well is imaged with a CCD camera Kits can also be used with Luminex 100 200 or Bio Rad Bio Plex dual laser flow based systems LIMITATIONS OF THE PROCEDURE e FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES e The kit should not be used beyond the expiration date on the kit label e Do not mix or substitute reagents with those from other lots or sources e If samples fall outside the dynami
13. r measurement It should be noted that many protocols for plasma preparation including procedures recommended by the Clinical Laboratory and Standards Institute CLSI result in incomplete removal of platelets from blood This will cause variable and irreproducible results for assays of factors contained in platelets and released by platelet activation Urine Aseptically collect the first urine of the day mid stream voided directly into a sterile container Centrifuge to remove particulate matter assay immediately or aliquot and store at lt 20 C Avoid repeated freeze thaw cycles Human Milk Assay immediately or aliquot and store samples at lt 20 C Avoid repeated freeze thaw cycles 4 For research use only Not for use in diagnostic procedures SAMPLE PREPARATION Cell culture supernate and urine samples require a 5 fold dilution A suggested 5 fold dilution is 50 uL of sample 200 uL of Calibrator Diluent RD6 49 diluted 2 1 Mix thoroughly Serum and platelet poor plasma samples require a 5 fold dilution A suggested 5 fold dilution is 50 uL of sample 200 uL of Calibrator Diluent RD6 49 Mix thoroughly Note Angiogenin serum and platelet poor plasma samples require an additional 10 fold dilution A suggested 10 fold dilution is 50 uL of diluted sample 450 uL of Calibrator Diluent RD6 49 to complete the 50 fold dilution Mix thoroughly Human milk samples require a 5 fold dilution A suggested 5 fold dilution is 5
14. r should be set at the low RP1 target value www RnDSystems com 7 ASSAY PROCEDURE Bring all reagents and samples to room temperature before use It is recommended that all samples and standards be assayed in duplicate Note Protect microparticles and Streptavidin PE from light at all times a 10 Prepare all reagents working standards and samples as directed in the previous sections Add 100 uL of Standard or sample per well Resuspend the diluted microparticle cocktail by inversion or vortexing Add 50 uL of the microparticle cocktail to each well of the microplate Securely cover with a foil plate sealer Incubate for 2 hours at room temperature on a horizontal orbital microplate shaker 0 12 orbit set at 800 50 rpm A plate layout is provided to record standards and samples assayed Using a magnetic device designed to accommodate a microplate wash by applying the magnet to the bottom of the microplate removing the liquid filling each well with Wash Buffer 100 uL and removing the liquid again Complete removal of liquid is essential for good performance Perform the wash procedure three times Note Refer to the magnetic device user manual for proper wash technique using a round bottom microplate Add 50 uL of diluted biotin antibody cocktail to each well Securely cover with a foil plate sealer and incubate for 1 hour at room temperature on the shaker set at 800 50 rpm Repeat the wash as
15. tive May turn yellow over time 641385 1 flat bottomed 96 well microplate used as a vessel for the assay Mixing Bottles 895505 2empty 8 mL bottles used for mixing microparticles with Microparticle Diluent Plate Sealers 640445 4adhesive foil strips Standard Value Card 749101 1 card listing the Standard reconstitution volume and working standard concentrations for this lot of base kit Provided this is within the expiration date of the kit Discard after use Use a fresh standard for each assay OTHER SUPPLIES REQUIRED e Luminex Performance Assay analyte specific kit s see Introduction on page 1 e Luminex MAGPIX Luminex 100 200 or Bio Rad Bio Plex analyzer with X Y platform e Hand held microplate magnet or platewasher with a magnetic platform e Pipettes and pipette tips e Deionized or distilled water e Multi channel pipette manifold dispenser or automated dispensing unit e 50 mL and 500 mL graduated cylinders e Horizontal orbital microplate shaker 0 12 orbit capable of maintaining a speed of 800 50 rpm e Microcentrifuge Polypropylene test tubes for dilution of standards and samples www RnDSystems com 3 PRECAUTIONS Calibrator Diluent RD6 49 contains sodium azide which may react with lead and copper plumbing to form explosive metallic azides Flush with large volumes of water during disposal Some components in this kit contain ProClin which may cause an allergic skin reaction Avo
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