Bio-rad Bio-Plex Pro™ Rat Cytokine, Chemokine, and Growth User Manual

Bio-Plex Pro™ Cytokine, Chemokine, and Growth Factor AssaysInstruction ManualFor technical support, call your local Bio-Rad office, or in the U.S.,

81. Plan Plate LayoutPrior to running the assay, determine the total number of wells in the experiment using the Plate Layout Template on page 40 or t

92. Prepare InstrumentStart up and calibrate the Bio-Plex® 100/200 or similar system with Bio-Plex Manager™ software prior to setting up the assay. T

103. Prepare Wash MethodBio-Plex Pro™ assays are compatible with both magnetic separation and vacuum filtration methods. However, for best results, we

114. Prepare StandardsGeneral Instructions n It is essential to reconstitute and dilute standards exactly as described in this section. Incorrect pre

12Assay Low RP1 (PMT) High RP1 (PMT)Human cytokines (group l, ll) •User validation required* Mouse cytokines (group l, ll, III) •User validation r

132. Add 500 μl of the appropriate diluent (see Table 5). Do not use assay buffer to reconstitute the standards.3. Gently vortex the reconsti

14Reconstituting Standards for Cross-Panel PlexingFollow these directions when mixing human or mouse cytokine and diabetes assays. Note that rat diabe

154. Add 150 μl of standard diluent to the remaining tubes, as shown in Figure 4.Fig. 4. S1 mixture and fourfold dilution series of diabetes and

165. Prepare SamplesGeneral guidelines for preparing different sample types are provided here. For more information, contact Bio-Rad Technical Support

172. For serum, allow blood to clot at room temperature for 30 to 45 min. For plasma, proceed directly to the centrifugation steps.3. Perform

Table of ContentsIntroduction 1Principle 2Kit Contents and Storage 4Recommended Materials 5Assay Workflow 6Important Considerations 7Detailed Instr

18LysatesThe Bio-Plex cell lysis kit is required for lysate preparation (available separately, catalog #171-304011 and #171-304012). Refer to bulletin

19For the mouse ICAM-1 assay, dilute serum or plasma 1:200 in Bio-Plex serum-based diluent, which is included in the Bio-Plex Pro high dilution reagen

203. Vortex the stock coupled beads at medium speed for 30 sec. Carefully open the cap and pipet any liquid trapped in the cap back into the tube.

21Preparing 1x coupled beads from 20x stock (includes 20% excess volume) Table 12. Premixed panel or one singleplex assay.Table 13. Mixing two singlep

227. Run AssayConsiderationsn Bring all assay components and samples to room temperature before usen Use calibrated pipets and pipet carefully, av

23Add Coupled Beads, Standards, and Samples1. Cover unused wells with sealing tape.2. Prewet the filter plate. Skip this step if using a flat-bottom

24Prepare and Add Detection AntibodiesInstructions are provided for diluting the detection antibodies to a 1x concentration. When mixing diabetes and

25 20x Detection Detection Antibody # of Wells Antibodies, µl Diluent, µl Total Volume, µl 96 150 2,850 3,000 48 75 1,425 1,500

268. Cover plate with a new sheet of sealing tape and protect from light with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 30 min at room

278. Cover plate with a new sheet of sealing tape and protect from light with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 10 min at roo

1IntroductionCytokines, chemokines, and growth factors are a diverse group of cell signaling proteins expressed and secreted by virtually all cell typ

288. Read PlateBio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay data acquisition and analysis. Instructions for Luminex xPONENT s

292. Click Select Analytes and create a new panel. Visually confirm the selected analytes and proceed to step 3. a. Click the Add Panel button

30Table 25. Bead regions for Bio-Plex Pro cytokine assays. 3. Click Format Plate and format the plate according to the plate layout created in Se

314. Click Enter Standards Info in the Protocol Settings bar. a. Enter the highest concentration of each analyte in the top row (labele

327. Click Run Protocol and confirm that the assay settings are correct. a. Refer to Table 24 for the recommended RP1 (PMT) setting. Protoco

33Reacquire DataIt is possible to acquire data from a well or plate a second time using the Rerun/Recovery mode located below Start in the Run Protoco

34Luminex xPONENT SoftwareAlthough guidelines are provided here, consult the xPONENT software manual for more details. Perform a system initialization

35Possible CausesHigh Inter-Assay CV Standards were not reconstituted consistently between assaysReconstituted standards anddiluted samples were not

36Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay componentsnot equilibrated to room temperature prior to

37Possible CausesLow Bead Count Miscalculation of bead dilutionBeads clumped in multiplexbead stock tubeVacuum on for too long whenaspirating buffe

2PrincipleTechnologyThe Bio-Plex® multiplex system is built upon the three core elements of xMAP technology:n Fluorescently dyed microspheres (also

38Possible CausesHigh Background Signal Incorrect buffer was used (for example, assay buffer used to dilute standards)Accidentally spiked blank w

39Possible SolutionsPipet carefully when adding standards, samples, detection antibodies, and streptavidin-PE, especially when using a multichannel pi

40Plate Layout Template

41Calculation WorksheetIf using either a premixed panel or one singleplex assay with 20x stocks of beadsand detection antibodies, follow these directi

42If mixing singleplex assays with 20x stocks of beads and detection antibodies, follow these directions. If using 10x stocks, divide by 10 instead of

43If mixing diabetes assays (20x bead and detection antibody stocks) with cytokine assays (10x stocks), follow these directions. Note: Refer to Tabl

442. Determine the volume of 1x diabetes and cytokine detection antibodies needed. a) Each well requires 25 μl of detection antibodies (1x): ______

45Safety ConsiderationsEye protection and gloves are recommended when using these products.Consult the MSDS for additional information. The Bio-Plex P

46Premixed All-In-One Multiplex Assays Catalog # Bio-Plex Pro Human Cytokine 8-Plex Panel, 1 x 96 M50-000007ABio-Plex Pro Human Cytokine 17-Plex Pan

Life ScienceGroup 10-0021 0213 Sig 121210014905 Rev D Bio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61

33Biomarker of InterestMagnetic BeadCapture AntibodyBiotinylated Detection AntibodyPhycoerythrin Fluorescent ReporterStreptavidinFig. 1. Bio-Plex sand

4 1 x 96-Well 10 x 96-Well Component Format Format Standard diluent* 10 ml 100 ml Sample diluent* 40 ml 80 ml Assay buffer 50 ml 500 ml

5Table 2. Recommended materials.ItemBio-Plex Pro Assays Quick Guide 4Bio-Plex® 200 system or Luminex system with HTFBio-Plex validation kit Run the

6 Prewet wells (for lter plate only)Add 50 μl 1x beads to wellsWash 2 x 100 μlAdd 50 μl standards, blank, samples, incubate at RT wi

7 Human, mouse, and rat diabetes 0 2 4 6 8 10 Mouse cytokine (group III) analytes (20x) Human and mouse cytokine (groups I, II) anal