Human High Sensitivity Cytokine Base Kit B
Contents
1. eene reete tte nananznnzenzan arana 6 INSIRUMENT SETTING ise ps ead 7 ASSAY PRGCEEDUURE 8 CALCULATION OF RESULTS e 9 CALIBRA TIONG 9 eel i C a a S 10 PEATE BAQA a NaS cae aces 12 MANUFACTURED AND DISTRIBUTED BV 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 Cvtokines are intercellular signaling proteins released from a wide varietv of cells and tissues They play an integral role in regulating growth and cellular proliferation as well as modulating host response to infection injury and inflammation Cytokines also influence reproduction and bone remodeling A large number of cytokines are pleiotropic and share similar functions In addition many cytokines influence the production of other cytokines Analysis an2. 0 d Flow rate 60 uL minute fast e f Doublet Discriminator gates at approximately 8000 and 16 500 9 Collect MFI Sample size 50 uL Note The CAL2 setting for the Bio Rad Bio Plex analyzer should be set at the low RP1 target value www RnDSystems com ASSAV PROCEDURE Bring all reagents and samples to room temperature before use It is recommended that all samples and standards be assaved in duplicate Note Protect microparticles and Streptavidin PE from light at all times NO 3 Ui g Prepare all reagents working standards and samples as directed in the previous sections Resuspend the diluted microparticle cocktail by inversion or vortexing Add 25 uL of the microparticle cocktail to each well of the microplate Add 100 uL of Standard or sample per well Securely cover with a foil plate sealer Incubate for 3 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
3. fall outside the dynamic range of the assay further dilute the samples with the appropriate Calibrator Diluent and repeat the assay 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 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 Only the analytes listed on the Standard Value Card can be measured with this base kit TECHNICAL HINTS When mixing or reconstituting protein solutions always avoid foaming To avoid cross contamination change pipette tips between additions of each standard level between sample additions and between
4. provided The volume of the Microparticle Concentrate listed in the table below is for each analyte e g if measuring a full plate of IL 1B and IL 6 add 50 uL of IL 1B Microparticle Concentrate and 50 uL of IL 6 Microparticle Concentrate to 2 5 mL of Diluent RD2 5 Number of Wells Used Microparticle Concentrate Diluent RD2 5 96 50 0 uL t 2 50 mL 72 37 5 ul 1 875 mL 48 25 0 ul 1 25 mL 24 12 5 uL 0 625 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 prior 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 one vial of the Diluent RD2 5 Mix gently 6 For research use only Not for use in diagnostic procedures INSTRUMENT SETTINGS Luminex MAGPIX analvzer a Assign the microparticle region for each analvte being measured see 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 microparticle region for each analyte being measured see page 1 b 50 events bead c Minimum events
5. reagent additions 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 894489 2 vials of recombinant human cytokines Human HS Cytokine Panel B in a buffered protein base with preservatives lyophilized Diluent RD2 5 895982 2 vials 6 mL vial of a buffered protein May be stored for up to 1 month at 2 8 C base with blue dye and preservatives Once diluted any unused microparticle cocktail must be discarded Calibrator Diluent 895119 21 ml of a buffered protein base with RD5K preservatives For cell culture supernate samples Calibrator Diluent 895098 21 ml ofa buffered protein base with RD6 65 preservatives For serum plasma samples May be stored for up 1 month at 2 8 C Streptavidin PE 895613 5 5 mL of streptavidin phycoerythrin conjugate with preservatives Wash Buffer Concentrate 895003 21mL of a 25 fold concentrated solution of buffered surfactant with preservative May turn yellow over time 641385 1flat bottomed 96 well mic
6. 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 in step 4 Add 50 uL of 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 rpm Repeat the wash as in step 4 Resuspend the microparticles by adding 100 uL of Wash Buffer to each well Incubate for 2 minutes at room temperature on the shaker set at 800 50 rpm Read within 90 minutes using a Luminex or Bio Rad Analyzer Serum and plasma samples require dilution See 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 Intensitv MFI Create a standard curve for each analvte bv reducing the data using computer software capable of generating a five parameter logistic 5 PL curve fit If 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 cytokines produced at R amp D Syst
7. D Imager Alternatively kits can be used with the Luminex 100 200 or Bio Rad Bio Plex dual laser flow based sorting 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 microparticle 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 LIMITATIONS OF THE PROCEDURE 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 Do not mix or substitute reagents with those from other lots or sources If samples
8. Magnetic Luminex Performance Assay Human High Sensitivity Cytokine Base Kit B Catalog Number LBHS000 For the simultaneous quantitative determination of multiple human cytokine concentrations in cell culture supernates serum and plasma Note IL 10 is no longer suitable for use in this assay Please use Human High Sensitivity Cytokine Panel A to assay IL 10 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 PAGE INFRODU GIO IN B a a d M 1 PRINCIPLE OF TIE AS SAN m a ccd cea 2 LIMITATIONS OF THE PROCEDURE tieniti otc at ccce sud Rte o Prae cR ne it c EAD up 2 LU i i aa e DRA RUE 2 MATERIALS PROVIDED amp STORAGE CONDITIONS cssssssssssscsssssssssssseessssscsssesesesssessssssssessesessessesessnsseecescseseseeses 3 OTHER SUPPLIES REQUIBEDL ii pacar accent aca inpar n a pa ob piis 3 iss V1 ei m M 9 4 SAMPLE COLLEC HON S S TO A GE tutu rere ek 4 SAMPLE PREPARATION 4 REAGENT PREPARATION tai sas 5 DILUTED MICROPARTICLE COCKTAIL PREPARATION s ssssssssssscssssssssscssssssssssessssssssssessssssssesssssessssssssssssessseceoes 6 DILUTED BIOTIN ANTIBODY COCKTAIL PREPARATION
9. NTINUED Recombinant mouse GM CSF IFN y IL 18 IL 2 IL 5 IL 6 IL 7 IL 10 IL 13 IL 15 IL 17A IL 17F IL 22 IL 31 IL 33 IL 328 TNF a Recombinant rat GM CSF IFN y IL 18 IL 2 IL 5 IL 6 IL 10 IL 13 IL 17F IL 22 IL 23 TNF a Recombinant porcine GM CSF IFN y IL 18 IL 2 IL 5 IL 6 IL 10 TNF a Recombinant cotton rat IFN y IL 18 IL 2 IL 5 IL 6 IL 10 IL 13 TNF a Recombinant feline GM CSF IFN y IL 18 IL 2 IL 5 IL 6 IL 10 TNF a Recombinant canine GM CSF IFN y IL 18 IL 2 IL 5 IL 6 IL 10 IL 13 IL 17 TNF a Other recombinants rhesus macaque IL 1B rhesus macaque IL 5 rhesus macaque IFN y rhesus macaque TNF a rabbit IL 2 rabbit IL 6 rabbit TNF a guinea pig 1 18 guinea pig IL 10 bovine IFN y bovine IL 2 bovine IL 5 bovine TNF a Recombinant human multiplex partners GM CSF IFN V IL 18 IL 2 IL 5 IL 6 IL 7 IL 13 IL 15 IL 17A IL 17F IL 22 IL 23 IL 31 IL 33 IL 36B TNF a www RnDSystems com PLATE LAVOUT Use this plate lavout to record sta 1000000002 XC XC XC XC 1000000002 400000000 eC X X X X X X X 2 400000000 CX XXX XXX 100000000 CX CX X XXX 100000000 400000000 400000000 NOTES www RnDSystems com NOTES All trademarks and registered trademarks are the property of their respective owners 2015 R amp D Systems Inc 07 13 752810 2 3 15 14 For research use onl
10. d quantification of cytokines in biological fluids has thus become increasingly important Methods such as bioassay enzyme linked immunosorbent assay ELISA intracellular staining ribonuclease protection assay RPA and polymerase chain reaction PCR have all been used for quantifying cytokines however each of these techniques has limitations associated with it These techniques are not capable of measuring multiple cytokines simultaneously in a limited sample volume Assessing the levels of multiple cytokines may be more revealing than analyzing a single protein Quantifying multiple cytokines on an individual level can be time consuming and expensive When combined with separately available analyte specific microparticle sets this kit is an excellent tool for simultaneously assessing the levels of multiple human cytokines in a single sample Any combination of the following microparticle sets are suitable for use with this base kit Analyte Catalog Number Micropartide Region GM CSF LHSCM215 29 IFN y LHSCM285 30 IL 1B LHSCM201 20 IL 2 LHSCM202 19 IL 5 LHSCM205 22 IL 6 LHSCM206 25 IL 7 LBHS207 33 IL 13 LBHS213 34 IL 15 LBHS247 35 IL 17A LBHS317 36 IL 17F LBHS1335 37 IL 22 LBHS5782 38 IL 23 LBHS1716 39 IL 31 LBHS2824 42 IL 33 LBHS3625 18 11 366 18 5 1099 15 LHSCM210 12 www RnDSvstems com 1 PRINCIPLE OF THE ASSAV Magnetic Luminex Performance Assay multiplex kits are designed for use with the Luminex MAGPIX CC
11. ems www RnDSystems com SPECIFICITV The assav was tested for cross reactivitv and interference with the following factors Less than 0 596 cross reactivitv and interference was observed unless otherwise noted on the analyte specific datasheet Recombinant human A 1BB Ligand Amphiregulin Angiogenin Angiopoietin 2 APRIL BAFF BLyS BDNF Cardiotropin 1 CD4 CD40 CD40 Ligand CNTF EDA A2 EDA Fas Ligand FGF acidic FGF basic FGF 4 FGF 5 FGF 6 G CSF GDNF GITR GITR Ligand GRO a GRO B GRO v HB EGF HGF IGF I IL 1a IL 1F7 FIL1 zeta IL 1ra IL 1RI IL 1 RII IL 2 Ra IL 3 IL 3 Ra IL 4 IL 4 Ra IL 5 Ra IL 6 Ra IL 7 Ra IL 8 IL 9 IL 10 IL 11 IL 12 p70 IL 12 p40 IL 17B IL 17C IL 17D IL 17E IL 18 IL 19 IL 20 IL 21 IL 22 Ral IL 23 R IL 24 IL 26 IL 27 IL 28A IFN A2 IL 29 IFN A1 IL 32a IL 32B IL 32y IL 33 Pro IL 36a IL 368 IL 36y IL 1F9 IL 36Ra IL 1F5 Leptin LIF LIF Ra LIGHT M CSF MCP 1 Midkine MIF MIP 1a MIP 1B B NGF NT 3 NT 4 Oncostatin M OSM Osteoprotegerin OX40 Ligand PD ECGF PDGF AA PDGF AB PDGF BB Pleiotrophin PTN RANTES SCF SLPI SMDF Lvmphotoxin a1 B2 Lymphotoxin 2 81 TGF a TGF B1 TGF B2 TGF B3 TGF B5 TGF RII TNF RI TNF RII TRAIL TRANCE TWEAK VEGF VEGI 10 For research use only Not for use in diagnostic procedures SPECIFICITY CO
12. of Wash Buffer Concentrate to deionized or distilled water to prepare 500 mL of Wash Buffer Standard Reconstitute the Standard Cocktail with Calibrator Diluent RD5K for cell culture supernate samples or Calibrator Diluent RD6 65 for serum plasma 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 polypropylene tubes Pipette 500 uL of the reconstituted Standard into the Standard 1 tube Pipette 300 uL of the appropriate Calibrator Diluent into the remaining tubes Use Standard 1 to produce a 4 fold dilution series below Refer to analyte specific datasheet 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 100 uL 100 uL 100 uL 100 uL 100 uL a JE E E 500 uL Std mmm 3 A B B B B B C CH EL EM Standard Cocktail Standard 1 Standard2 Standard3 Standard4 Standard5 Standard6 Standard 7 www RnDSvstems com DILUTED MICROPARTICLE COCKTAIL PREPARATION 1 Centrifuge each Microparticle Concentrate vial for 30 seconds at 1000 x g prior to removing the cap 2 Gentiv vortex the vials to resuspend the microparticles taking precautions not to invert the vials 3 Dilute the Microparticle Concentrates in the mixing bottle
13. or to use SAMPLE COLLECTION amp 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 x 20 C Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA or heparin as an anticoagulant Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection 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 Hemolyzed icteric and lipemic sample are not suitable for use in this assay SAMPLE PREPARATION Serum and plasma samples require a 2 fold dilution A suggested 2 fold dilution is 150 uL of sample 150 uL of Calibrator Diluent RD6 65 Mix thoroughly 4 For research use only Not for use in diagnostic procedures 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
14. roplate used as a vessel for the assay Mixing Bottles 895505 2 8 mL bottles used for mixing micropartides with Microparticle Diluent Plate Sealers 640445 6 adhesive foil Gadhesive foll strips 0 Standard Value Card 749810 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 500 mL graduated cylinder 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 e Human HS Cytokine Panel B Controls R amp D Systems Catalog f QC19 optional www RnDSystems com 3 PRECAUTIONS Some components in this kit contain ProClin which may cause an allergic skin reaction Avoid breathing mist Wear protective gloves clothing eye and face protection Wash hands thoroughly after handling Please refer to the MSDS on our website pri
15. y Not for use in diagnostic procedures
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