Bio-rad Human Inflammation Assays Manuel d'utilisateur

Bio-Plex Pro™ Human Inflammation AssaysInstruction ManualFor technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723.

8 1. Plan Plate LayoutDetermine the total number of wells in the experiment using the Plate Layout Template on page 34 or the Plate Formatting tab

9 2. Prepare InstrumentThese directions are specific for the Bio-Plex® 100/200 reader. To prepare either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader,

10 Calibrate System 1. Select Calibrate and confirm that the default values for CAL1 and CAL2 are the same as the values printed on the bott

11 4. Preparing Buffer and DiluentPrepare Wash Buffer 1. Bring the 10x stock solution to room temperature. 2. If crystals are present, ensure

12 Reconstitute Standards and Quality ControlsThis procedure prepares enough standard to run each dilution in duplicate. Note: The appearance

13 2. Add 150 µl of the appropriate diluent to tubes S2–S8 (Figure 3).3. Vortex reconstituted standards at medium speed for 5 sec before

14 Serum and Plasma Note: If using plasma, EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma, while compatible with Bio-Ple

3. We recommend testing undiluted samples first. If high levels of analyte are expected, samples can be further diluted in culture medium. Rarely wo

16 5. Perform either of the following to remove insoluble cellular particulates: n Centrifuge the cell lysate solution at 4,500 x g for 20 mi

Lavage, Sputum, and Other Biological Fluid Samples Keep all samples on ice until ready for use. The appropriate sample dilution factor should be optim

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

18 3. Vortex the 10x stock of coupled beads at medium speed for 30 sec. Carefully open the cap and pipet any liquid trapped in the cap back in

19 n Use calibrated pipets and pipet carefully, avoiding bubblesn Pay close attention to vortexing, shaking, and incubation instructions. Devia

20 Prepare and Add Detection Antibodies1. While the samples are incubating use Tables 9 and 10 or the Calculation Worksheet on pages 35–36 to ca

21 7. Vortex the diluted (1x) detection antibodies at medium speed for 5 sec. Transfer into a reagent reservoir and dispense 25 µl to each well

22 8. 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 R

23 Bio-Plex Manager software versions 6.0 and higher contain protocols for most Bio-Plex® assays. Choose from available protocols or create a new p

24 Table 13. Bead regions for the human inflammation panel.TargetBead RegionTarget Bead RegionTarget Bead RegionAPRIL/TNFSF13 42 IL-11 39 LIGHT/TNFS

25 b. Enter a dilution factor of 3 and click Calculate. The concentrations for each standard point will be populated for all analytes in th

26 6. Click Enter Sample Info and enter sample information and the appropriate dilution factor.7. Click Run Protocol and confirm that the ass

27 Reacquire 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 Pro

IntroductionCytokines are low molecular weight proteins that are generated mostly by immune cells and in turn play crucial roles in a cascade of vascu

28 Removing OutliersOutliers are identified as standard data points that do not meet accuracy or precision requirements and should be considered inv

29 5. Analysis type: Quantitative, 5PL Curve Fit.6. Number of standards: 8.Select Analytes to set up the panel.1. Enter pg/ml in the Units fie

30 Possible CausesHigh Inter-Assay CV Standards and controls were not reconstituted consistently between assaysReconstituted standards, controls,

31 Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay components not equilibrated to room temperature pri

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

33 Possible CausesHigh Background Signal Incorrect buffer was used (for example, assay buffer used to dilute standards)Accidentally spiked blan

34 34 Possible SolutionsPipet carefully when adding standards, samples, detection antibodies, and streptavidin-PE, especially when using a multi

35 Plate Layout Template

36 Calculation WorksheetIf using either a premixed panel or one singleplex assay, follow these directions.Plan the plate layout and enter the numb

37 If mixing singleplex assays, follow these directions.Enter the number of wells to be used in the assay:_______ 11. Determine the volume of 1x

PrincipleTechnologyThe Bio-Plex® multiplex system is built upon the three core elements of xMAP technology:n Fluorescently dyed magnetic microsphere

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

39 Ordering InformationDetailed ordering information can be found at www.bio-rad.com/bio-plex.Bio-Plex Express Assays (You Mix) Fast and economica

Life ScienceGroup Sig 121310044282 Rev A US/EGBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 A

3 Fig. 1. Bio-Plex sandwich immunoassay. Data Acquisition and AnalysisData from the reactions are acquired using a Bio-Plex system or similar Lumin

4 Kit Contents and StorageReagents SuppliedBio-Plex Pro™ human inflammation assays are available in a convenient all-in-one kit format that includes

5 ItemBio-Plex Pro Inflammation Assays Quick GuideBio-Plex® MAGPIX™, Bio-Plex Manager™Bio-Plex validation kit Note: Run the validation kit monthly

6 Prewet wells (for lter plate only)Add 50 μl 1x beads to wellsWash 2 x 100 μlAdd 50 μl standards, samples, controls; incubate on shaker at 850 r

7 lmportant ConsiderationsInstruments and Software The Bio-Plex Pro™ assays described in this manual are compatible with all currently available Lu