Bio-rad Human Metabolic and Hormone Assays User Manual

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

8lmportant ConsiderationsInstruments and Software The assays described in this manual are compatible with all currently available Luminex-based life s

91. Plan Plate LayoutDetermine the total number of wells in the experiment using the Plate Layout Template on page 31 or the Plate Formatting tab in B

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

112. 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

12Setting Up the Bio-Plex Handheld Magnetic Washer Place an empty flat bottom plate on the magnetic washer by sliding it under the retaining clips. Pus

13Dilution of Standard (1:3 Serial Dilution)This preparation provides sufficient volume to run duplicate standard dilution curves. Ensure that each new

145. Prepare SamplesNote: Most of the circulating IGF-1 and IGF-2 are complexed with the IGFBPs. An extraction step to separate the complexes is requi

15Protease InhibitorsIn general, these biomarkers are detectable in serum or plasma without using protease inhibitors. Users may choose to add proteas

16Cell 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 medi

175. After the incubation, centrifuge the extraction tubes in a microcentrifuge at 8,400 x g for 10 min (±1 min).6. Add 50 µl neutralization

Table of ContentsIntroduction 1Principle 3Kit Contents and Storage 5Recommended Materials 6Assay Workflow 7lmportant Considerations 8Detailed Inst

18Panel Sample DilutionVolume of Sample, µlVolume of Extraction BufferVolume of Neutralization BufferVolume of Sample Dilution Buffer 2IGF 1:30 20 180

19Considerations When Using a Vacuum Manifoldn After each incubation, place the filter plate on a calibrated vacuum apparatus and remove the liquid b

8. Add 20 µl diluted SA-PE to the required plate wells.9. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.10. Wash the plate

21To create a new protocol, select File, then New from the main menu. Locate and follow the steps under Protocol Settings.1. Click Describe Protoco

223. Click Format Plate and format the plate according to the plate layout template (located on page 31) created for the assay. To modify the plate

235. Click Enter Controls Info. a. For user-specified controls, select an analyte from the dropdown menu, then enter a description and concentrat

242. Click Run Protocol — on the pop-up screen, select Load Plate and click OK to start acquiring data.3. Use the Wash Between Plates comman

25Previous Versions of Bio-Plex Manager SoftwareFor instructions on using previous versions of Bio-Plex Manager software, please contact Bio-Rad Techn

26Possible CausesHigh Inter-Assay CV Standards were not reconstituted consistently between assaysReconstituted standards and diluted samples were no

27Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay componentsnot equilibrated to room temperature prior to

1IntroductionInsulin-like growth factors (IGFs), along with their binding proteins (IGFBPs), have been shown to play key roles in cell growth, organog

28Possible CausesLow Bead CountVacuum on for too long whenaspirating buffer from wells Reader is clogged Low Signal or Poor SensitivityStandards reco

2929Possible CausesPoor Recovery Expired Bio-Plex reagents were used Incorrect amounts of components were addedMicroplate shaker set to an

3030Possible SolutionsIf samples contain little or no analyte, negative values observed may be due to statistical variation. If assay drift is suspect

31Plate Layout Template31

32Safety ConsiderationsEye protection and gloves are recommended when using these products.Consult the MSDS for additional information. The Bio-Plex P

33Ordering 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 121310042015 Rev A US/EGBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 A

2ReferencesHjortebjerg R and Frystyk J (2013). Determination of IGFs and their binding proteins. Best Pract Res Clin Endocrinol Metab 27, 771–781.Juul

3PrincipleTechnologyThe Bio-Plex® multiplex system is built upon the three core elements of xMAP technology:n Fluorescently dyed magnetic microspher

4Fig. 1. Bio-Plex sandwich immunoassay. Data Acquisition and AnalysisData from the reactions are acquired using a Bio-Plex system or similar Luminex-b

5Kit Contents and StorageThe Bio-Plex Pro™ RBM IGF and IGFBP assays are available in a convenient kit format that includes assay, reagent, and diluent

6ItemBio-Plex® 200 system or Luminex system with HTFBio-Plex validation kit Note: Run the validation kit monthly to ensure optimal performance of fluid

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