Bio-rad Apoptosis Assays Manuel d'utilisateur

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

81. Plan Plate LayoutDetermine the total number of wells in the experiment using the Plate Layout Template on page 32 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

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

11Setting 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

122. Bring the 10x assay buffer to room temperature (RT). a. Mix by inversion to ensure all salts are in solution. b. Prepare 1x assay buffe

13Fig. 3. Preparing a threefold dilution series with a single reconstituted standard.5. Prepare SamplesThe kit has sufficient reagents to run 19 fracti

14 b. Homogenizer — Bio-Gen Pro200 (cat #01-01200) with a 5 mm generator (cat #02-05075) or equivalent. c. Dounce homogenizer — Fisher (cat #06-434

15homogenize the minced tissue for approximately 5 sec on medium power. Homogenized tissue samples should be free of large tissue fragments. If large

166. Aliquot samples at appropriate volumes into labeled tubes and store at –80°C. Prepare an aliquot for protein determination. Cytosolic fractions

172. Prechill all tubes and keep samples on ice during sample preparation. LDB additions and homogenization of samples are summarized in Table 6.3.

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

18 c. Cultured Cells i. Collect the cells (5 x 106 to 2 x 107) by centrifugation at 4°C for 5 min at 500 x g. Discard supernatant. ii. W

Table 7. Summary of wash options and protocols. After each assay step, select the appropriate Bio-Plex Pro wash station program or perform the appropr

207. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do not aspirate after incubation.8. Prepare the required dilution of streptav

21Prepare Protocol in Bio-Plex Manager Software Version 6.0 and HigherThe protocol should be prepared in advance so that the plate is read as soon as

22 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 rename the

23 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 beads

24 a. Confirm that data acquisition is set to 50 beads per region. b. In Bio-Plex Manager software prior to 6.1, go to Advanced Settings, confirm

25Data AnalysisQuality ControlsIf the quality controls were run in the assay plate, open the results (.rbx) file, click Report Table, and locate the co

26Luminex xPONENT SoftwareLuminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPONENT

27Possible CausesHigh Inter-Assay CV Standards were not reconstituted consistently between assaysReconstituted standards and diluted samples were no

1IntroductionApoptosis refers to a genetically controlled process by which cells die following a programmed physiological state or pathological condit

28Possible CausesHigh Intra-Assay CV Improper pipetting technique Reagents and assay componentsnot equilibrated to room temperature prior to

29Possible CausesLow Bead CountVacuum on for too long whenaspirating buffer from wells Reader is clogged Low Signal or Poor SensitivityStandards reco

30Possible CausesPoor Recovery Expired Bio-Plex reagents were used Incorrect amounts of components were addedMicroplate shaker set to an in

31Possible SolutionsIf samples contain little or no analyte, negative values observed may be due to statistical variation. If assay drift is suspected

Plate Layout Template32

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

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

Life ScienceGroup Sig 121210033631 Rev A Bio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria

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 apoptosis assays are available in a convenient kit format that includes assay, reagent, and diluent com

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 Reconstitute lyophilized reagents, dilute assaybuffer to 1x, prepare standards and samplesAdd 10 µl blocking buffer to all wellsAdd 30 μl standards,

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