Bio-rad Human Metabolic and Hormone Assays User Manual

Bio-Plex Pro™ RBM Metabolic and Hormone AssaysInstruction ManualFor technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424

81. Plan Plate LayoutDetermine the total number of wells in the experiment using the Plate Layout Template on page 29 or the Plate Formatting tab in B

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

102. Select OK and follow the software prompts for step-by-step instructions for CAL1 and CAL2 calibration.Note: In Bio-Plex Manager version 6.1 and

11Setting Up a Vacuum Manifold Calibrate the vacuum manifold by placing a standard 96-well flat bottom plate on the unit and adjusting the pressure to

12 Reconstituted Standard 100 100 100 100 100 100 100 100 S1 S2 S3 S4 S5 S6 S7 S8 BlankStandard Diluent, µl 150 50 50 50 50 50 50 50 Trans

135. Prepare SamplesThe kit has sufficient reagents to run 37 samples in duplicate for a full plate analysis. General guidelines for preparing samples

14Protease InhibitorsIn general, these biomarkers are detectable in serum or plasma without using protease inhibitors. Users may choose to add proteas

15Cell culture media1. Collect cell culture supernatants and centrifuge at 1,000 x g for 15 min at 4°C. For cell lines cultured in serum-free media,

166. Run the AssayConsiderationsn Bring all assay components and samples to room temperature before usen Use calibrated pipets and pipet carefully

17Assay Protocol: Dispensing of Reagents1. Add 10 µl blocker to all wells of the plate.2. Add 30 µl the standard, control, or sample to the appr

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

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

d. Highlight analytes from the Available list (left) and move to the Selected list (right) using the Add button. To move all analytes at once, simp

203. Click Format Plate and format the plate according to the plate layout template (located on page 29) created for the assay. To modify the plate

21Table 9. Read the plate using the appropriate instrument settings. a. Confirm that data acquisition is set to 50 beads per region. b. In Bio-Pl

22Data AnalysisQuality ControlsIf the quality controls were run in the assay plate, open the results (.rbx) file, click Report Table, and locate the co

23Luminex xPONENT SoftwareLuminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPONENT

24Possible CausesHigh Inter-Assay CV Standards were not reconstituted consistently between assaysReconstituted standards and diluted samples were no

25Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay componentsnot equilibrated to room temperature prior to

26Possible CausesLow Bead CountVacuum on for too long whenaspirating buffer from wells Reader is clogged Low Signal or Poor SensitivityStandards reco

2727Possible CausesPoor Recovery Expired Bio-Plex reagents were used Incorrect amounts of components were addedMicroplate shaker set to an

1IntroductionApproximately 65% of individuals with diabetes die from heart disease and stroke. Individuals with type 2 diabetes also have an increased

2828Possible SolutionsIf samples contain little or no analyte, negative values observed may be due to statistical variation. If assay drift is suspect

29Plate Layout Template29

30Safety ConsiderationsEye protection and gloves are recommended when using these products.Consult the MSDS for additional information. The Bio-Plex P

31Ordering InformationDetailed ordering information can be found at www.bio-rad.com/bio-plex.Catalog # Premixed 1 x 96-Well All-In-One Multiplex Kit

Life ScienceGroup Sig 121310041818 Rev A US/EGBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 A

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

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

4Kit Contents and StorageThe Bio-Plex Pro™ RBM metabolic and hormone assays are available in a convenient kit format that includes assay, reagent, and

5ItemBio-Plex® 200 system or Luminex system with HTFBio-Plex validation kit Note: Run the validation kit monthly to ensure optimal performance of fluid

6 Prepare samples, reconstitute lyophilized reagents, dilute assay buffer to 1x, prepare standardsAdd 10 µl blocking buffer to all wellsAdd 30 μl stan

7lmportant ConsiderationsInstruments and Software The assays described in this manual are compatible with all currently available Luminex-based life s