Bio-rad Bio-Plex Pro™ Human Th17 Cytokine Assays User Manual

Bio-Plex Pro™ Human Th17 Cytokine AssaysInstruction ManualFor technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723

8 1. Plan Plate LayoutPrior to running the assay, determine the total number of wells in the experiment using the plate layout template on page 34,

9 2. Prepare InstrumentStart up and calibrate the Bio-Plex® system with Bio-Plex Manager™ software prior to setting up the assay. The calibration k

10 3. Prepare Wash MethodThere are two methods of performing wash steps in the assay procedure: magnetic separation and vacuum filtration. Compatibl

11 4. Prepare Standards and ControlsGeneral Instructions • Itisessentialtopreparestandardsandqualitycontrols(ifincluded)exactly as des

12 Reconstitute Standards and Quality ControlsThis procedure prepares enough standard to run each dilution in duplicate. 1. Gently tap the

13 4. Use a new pipet tip to transfer 50 µl from the S2 tube to the S3 tube. Vortex for 5 sec.5. Continue with 1:4 (fourfold) serial d

14 Sample PreparationSerum and Plasma EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma, while compatible with Bio-Plex Pro

Cell Culture Supernatant 1. Collect supernatants and centrifuge at 1,000 x g for 15 min at 4°C. For cell lines cultured in serum-free culture

16 2. Determine the total protein concentration of the lysate. It may be necessary to test lyse your samples with different volumes of lysing so

Tables 6–7 summarize the volumes required to prepare 1x beads from a 20x stock. Also shown are volumes for preparing 1x beads when mixing two 20x sto

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

18 Table 8. Detailed wash and incubation instructions.Add Coupled Beads, Samples, Standards, Blank, and Controls1. Cover unused wells of the assa

19 3. Vortex the diluted (1x) beads for 30 sec at medium speed. Pour into a reagent reservoir and transfer 50 µl to each well of the assay plate

20 Tables 9–10 summarize the volumes required to prepare 1x detection antibodies from a single 20x stock. Also shown are volumes to prepare 1x anti

21 Prepare and Add Streptavidin-PE (SA-PE)1. While detection antibodies are incubating, use Table 11 or the Calculation Worksheet on page 35 to

22 9. After the streptavidin-PE incubation step, slowly remove and discard the sealing tape.10. Wash the wells three times with 100 µl of wash

23 1. Describe Protocol and enter information about the assay (optional).2. Select Analytes and create a new panel. Visually confirm the select

24 e. If some of the analytes need to be removed from the Selected list, highlight them and select Remove. If desired, it is possible to

25 4. Enter Standards Info into Bio-Plex Manager. a. Enter the highest concentration of each analyte in the top row (labeled S1) of the

26 Acquire Data1. Shake the assay plate at 850 ±50 rpm for 30 sec, and visually inspect the plate to ensure that the assay wells are filled wi

27 Data AnalysisQuality ControlsIf the quality controls were run in the assay plate, open the results (.rbx) file, click on Report Table, and locate

IntroductionBio-Plex Pro™ Human Th17 Cytokine Assays Bio-Plex Pro human Th17 cytokine assays are designed to meet the needs of the most demanding prec

28 Luminex xPONENT SoftwareLuminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPON

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

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

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

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

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

34 Plate Layout Template

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

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

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

PrincipleTechnologyThe Bio-Plex® suspension array system is built around the three core elements of xMAP technology:• Fluorescentlydyedmicrosphere

38 Ordering InformationBio-Plex® x-Plex™ Assays (We Mix)Premium, custom assay service using the Bio-Plex Assay Builder, www.bio-rad.com/bio-plex/a

39

Life ScienceGroup Sig 121110023381 Rev A Bio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austri

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

4 Kit Contents and StorageReagents SuppliedBio-Plex Pro™ human Th17 cytokine assays are available in a convenient all-in-one kit format that includ

5 ItemBio-Plex Pro Assays Quick Guide 1Bio-Plex® 200 system or Luminex system with HTFBio-Plex validation kit Note: the validation kit should be r

6 Prewet wells (for lter plate only)Add beadsWash 2xAdd standards, samples, controls, 1 hrWash 3xAdd detection antibody, 30 minWash 3xAdd strept

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