Bio-rad Human MMP and TIMP Assays User Manual

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

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 33

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

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

12 Sample Type Diluent for Standard and Controls* Add BSA Serum and plasma Diluent HD None Culture media, with serum Culture media None Cul

13 Prepare the Standard Dilution SeriesThe following procedure produces an eight-point standard curve with a threefold dilution between each

14 6. Prepare SamplesThese assays are designed to quantitate classes and subclasses of immunoglobulins in serum, plasma, and cell culture media. Fo

3. Perform centrifugation at 1,000 x g for 15 min at 4°C and transfer the serum or plasma to a clean polypropylene tube.4. To completely remove

16 7. Prepare Coupled Beads1. Use Table 6 or the Calculation Worksheet on page 34 to calculate the volume of coupled beads and assay buffer need

8. Run AssayConsiderationsn Bring all buffers, diluents, diluted standards, diluted coupled beads, and samples to room temperature before usen Use

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

18 Add Coupled Beads, Samples, Standards, Blank, and Controls1. Cover unused wells of the assay plate with sealing tape.2. Prewet the filter pla

19 4. Dilute detection antibodies to 1x by pipetting the required volume into the 15 ml tube. Vortex. Each well of the assay requires 1.25 

20 4. Dilute SA-PE to 1x by pipetting the required volume into the 15 ml tube. Vortex and protect from light until ready to use. Each well

21 9. Read PlateBio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay data acquisition and analysis. Instructions for Luminex xPONE

22 b. Click the Add button. Enter the bead region number and name for the first analyte. Click Add Continue to repeat for each analyte in the as

23 Fig. 4. Plate formatting.4. Click Enter Standards Info. a. Enter the highest concentration and units of each analyte in the top row (labe

24 Instrument RP1 (PMT) DD Gates Bead Events Bio-Plex 100, 200* Low 5,000 (low), 25,000 (high) 50 Bio-Plex 3D* Standard Select MagPlex b

25 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 with

26 Data AnalysisControlsIf the controls were run in the assay plate, open the results (.rbx) file, click on Report Table, and locate the control we

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

IntroductionBio-Plex Pro™ Human TIMP Assays The matrix metalloproteinases (MMPs) are a family of zinc proteases with essential roles in breaking down

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

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

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

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

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

33 Plate Layout Template

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

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

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

Life ScienceGroup Sig 121310041640 Rev A Bio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Au

PrincipleTechnologyThe Bio-Plex® suspension array system is built upon the three core elements of xMAP technology:n Fluorescently dyed microspheres

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™ TIMP assays are available in a convenient all-in-one kit format that includes assay, reage

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

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

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