Bio-Plex Pro RBM Apoptosis Assays ™ Instruction Manual For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723. For research use only. Not for diagnostic procedures.
Table of Contents Introduction 1 Principle 2 Kit Contents and Storage 4 Recommended Materials 5 Assay Workflow 6 lmportant Considerations 7 Detailed Instructions 7 1. Plan Plate Layout 8 2. Prepare Instrument 9 3. Prepare Wash Method 10 4. Prepare Reagents 11 5. Prepare Samples 13 6. Run the Assay 18 7.
Introduction Apoptosis refers to a genetically controlled process by which cells die following a programmed physiological state or pathological condition. The balance between pro- and anti-apoptotic processes is delicate, and dysregulation is observed in a wide range of pathological conditions (Rutledge 2002). Excessive apoptosis is implicated in neurodegenerative and autoimmune diseases, myocardial infarction, stroke, and viral infection.
Principle Technology The Bio-Plex® multiplex system is built upon the three core elements of xMAP technology: n n n Fluorescently dyed magnetic microspheres (also called beads), each with a distinct color code or spectral address to permit discrimination of individual tests within a multiplex suspension.
Biomarker of Interest Streptavidin Magnetic Bead Capture Antibody Biotinylated Detection Antibody Phycoerythrin Fluorescent Reporter Fig. 1. Bio-Plex sandwich immunoassay. Data Acquisition and Analysis Data from the reactions are acquired using a Bio-Plex system or similar Luminex-based reader.
Kit Contents and Storage The Bio-Plex Pro™ RBM apoptosis assays are available in a convenient kit format that includes assay, reagent, and diluent components in a single box (Table 1). All other recommended materials are listed in Table 2. Table 1. Contents of 1 x 96-well kits. Component Quantity Volume Volume after Reconstitution or Dilution Capture beads (1x) 1 tube Detection antibodies 1 vial Lyophilized 1.4 ml 4.
Table 2. Recommended materials.
Assay Workflow Reconstitute lyophilized reagents, dilute assay buffer to 1x, prepare standards and samples Add 10 µl blocking buffer to all wells Add 30 μl standards, blank, samples, and controls to appropriate wells Add 10 μl 1x capture beads per well. Incubate at 850 ± 50 rpm for 1 hr at RT Wash 3x with 100 µl assay buffer (1x) Add 40 μl reconstituted detection antibodies. Incubate at 850 ± 50 rpm for 1 hr at RT Do not aspirate after incubation Add 20 μl 1x streptavidin-PE.
lmportant Considerations Instruments and Software The assays described in this manual are compatible with all currently available Luminex-based life science research instruments. Assays can be read and analyzed with either Bio-Plex Manager™ software or Luminex xPONENT software. Assay Procedures Pay close attention to vortexing, shaking, and incubation times and to Bio-Plex® reader PMT (RP1) setting, as these have been optimized specifically for each assay panel.
1. Plan Plate Layout Determine the total number of wells in the experiment using the Plate Layout Template on page 32 or the Plate Formatting tab in Bio-Plex Manager™ software. A suggested plate layout is shown in Figure 2, with all conditions in duplicate. 1. Assign standards to columns 1 and 2, with the highest concentration in row A and the lowest concentration in row H. 2. Assign the blank to wells A3 and A4.
2. Prepare Instrument These directions are specific for the Bio-Plex® 100/200 reader. To prepare either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader, consult their respective user manuals. Start up and calibrate the Bio-Plex® system with Bio-Plex Manager™ software prior to setting up the assay. The calibration kit should be run daily or before each use of the instrument to standardize the fluorescent signal. For instructions on using other xMAP system software packages, contact Bio-Rad Technical Support.
Calibrate 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 Bio-Plex calibration beads. Use the Bio-Plex system low RP1 target value. 2. 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 higher, startup, warm up, and calibration can be performed together by selecting the “Start up and calibrate” icon. 3.
Setting Up the Bio-Plex Handheld Magnetic Washer Place an empty flat bottom plate on the magnetic washer by sliding it under the retaining clips. Push the clips inward to secure the plate. Make sure the plate is held securely. If needed, the clips can be adjusted for height and tension. For detailed instructions, refer to the user guide (bulletin #10023087).
2. Bring the 10x assay buffer to room temperature (RT). a. M ix by inversion to ensure all salts are in solution. b. Prepare 1x assay buffer — dilute 1 part 10x assay buffer (60 ml) with 9 parts of dH20 (540 ml). Test Sample Preparation Thaw and dilute samples within 1 hr of use. Remove any particulates by centrifugation or filtration. Avoid multiple freeze and thaw cycles. Dilution of Standard (1:3 Serial Dilution) This preparation provides sufficient volume to run duplicate standard dilution curves.
0 Reconstituted Standard 50 S1 50 50 50 50 50 50 Transfer Volume, µl 100 100 100 100 100 100 100 100 S2 S3 S4 S5 S6 S7 S8 Blank Standard Diluent, µl Fig. 3. Preparing a threefold dilution series with a single reconstituted standard. 5. Prepare Samples The kit has sufficient reagents to run 19 fractionated samples (cytosolic and nuclear + mitochondrial fractions) in duplicate or 38 total homogenates in duplicate.
b. Homogenizer — Bio-Gen Pro200 (cat #01-01200) with a 5 mm generator (cat #02-05075) or equivalent. c. D ounce homogenizer — Fisher (cat #06-434) using tight pestle, or equivalent. Preparation of Cytosolic and Nuclear + Mitochondrial (N + M) Fractions Cytosolic Fraction 1. Prepare the cytosolic extraction buffer (CEB) and lysate dilution buffer (LDB) by adding 1 tablet each of the protease inhibitors listed above to 10 ml of each buffer.
homogenize the minced tissue for approximately 5 sec on medium power. Homogenized tissue samples should be free of large tissue fragments. If large fragments are visible in the sample, homogenize for an additional 5 sec. Any large fragments remaining after the additional 5 sec homogenization step should be removed before proceeding. Incubate processed samples on ice for 10 min. After incubation, transfer the sample to a prechilled 1.5 ml microcentrifuge tube. b.
6. Aliquot samples at appropriate volumes into labeled tubes and store at –80°C. Prepare an aliquot for protein determination. Cytosolic fractions may be stored on ice while the nuclear + mitochondrial fractions are prepared. Nuclear + Mitochondrial Fraction 7. Wash the pellet from the cytosolic preparation by resuspending in 750 µl of CEB and centrifuge at 4°C for 10 min at 10,000 x g. 8. Aspirate and discard the supernatant from the tube, being careful not to disturb the pellet. 9.
2. Prechill all tubes and keep samples on ice during sample preparation. LDB additions and homogenization of samples are summarized in Table 6. Table 6. Summary of homogenization methods for total extracts.
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. Wash the cells in 1–2 ml of ice-cold PBS. Centrifuge at 4°C for 5 min at 500 x g. Discard supernatant. iii. Add 750 µl LDB to the pellet, resuspend the cells by pipetting up and down 10–15x, and vortex for 5 sec. iv. Incubate the suspension on ice for 30 min. v. Homogenize using with a Dounce homogenizer. vi. Transfer the homogenate to a 1.
Table 7. Summary of wash options and protocols. After each assay step, select the appropriate Bio-Plex Pro wash station program or perform the appropriate manual wash step as summarized below.
7. 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 streptavidin-PE (SA-PE) as outlined in Table 8. Note: Volumes in the table are for an entire 96-well plate. Smaller volumes can be prepared, provided that dilution ratios are maintained. Table 8. SA-PE dilution. SA-PE Dilution Volume of SA-PE, µl Volume of 1x Assay Buffer, µl Total Volume, µl 1:10 225 2,025 2,250 9.
Prepare Protocol in Bio-Plex Manager Software Version 6.0 and Higher The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete. A protocol file specifies the analytes used in the reading, the plate wells to be read, sample information, the values of standards and controls, and instrument settings. Protocols may be obtained from within Bio-Plex Manager software version 6.1 or created from the File menu.
e. I f 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 panel by clicking on Rename Panel and entering a new panel name. Table 9. Bead regions for Bio-Plex Pro RBM apoptosis assays. Panel 1 Bak (74) Bax (27) Lamin B (14) Smac (19) Panel 2 Bad (73) Bcl-2/Bax (42) Bcl-xL (22) Bim (12) Active caspase (57) Bcl-xL/ Bak (47) Mcl-1/Bak (54) Survivin (20) Panel 3 Mcl-1 (18) 3.
4. Click Enter Standards Info in the Protocol Settings bar. a. Enter the highest concentration of each analyte in the top row (labeled S1) of the table. S1 concentration information is listed in the product data sheet. b. Enter a dilution factor of 3 and click Calculate. The concentrations for each standard point will be populated for all analytes in the table. c. Optional: enter the lot number of the vial of standards into the Standard Lot box and click Save. 5. Click Enter Controls Info. a.
a. Confirm that data acquisition is set to 50 beads per region. b. I n Bio-Plex Manager software prior to 6.1, go to Advanced Settings, confirm that the bead map is set to 100 region, the sample size is set to 50 µl, and the DD gates are set to 5,000 (Low) and 25,000 (High). In Bio-Plex Manager software versions 4.0, 4.1, 4.1.1, and 5.0, check Override Gates and set the DD gate values as indicated. Select Start, name and save the .rbx file, and begin data acquisition.
Data Analysis Quality Controls If the quality controls were run in the assay plate, open the results (.rbx) file, click Report Table, and locate the control wells. Compare the observed concentrations against the lot-specific control ranges in the product data sheet. Note: Expected control ranges are provided for reference and should be used as general guidelines. Actual results may vary for some operators.
Luminex xPONENT Software Luminex xPONENT software may be used to analyze Bio-Plex assays. Although guidelines are provided here, consult the xPONENT software manual for more details. Perform a system initialization with Luminex’s calibration and performance verification kit, as directed by Luminex. Select Batches to set up the protocol and follow the information under Settings. Note: The instrument settings described below apply to Luminex 100/200 and FLEXMAP 3D or Bio-Plex 3D instruments.
Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio-Plex Pro™ RBM assays. If you experience any of the problems listed below, review the possible causes and solutions provided. Poor assay performance may also be due to the Bio-Plex® suspension array reader. To eliminate this possibility, use the validation kit to validate all the key functions of the array reader and to assist in determining whether or not the array reader is functioning properly.
Possible Causes Possible Solutions High Intra-Assay CV Improper pipetting technique Pipet carefully when adding standards, samples, detection antibodies, and streptavidin-PE, especially when using a multichannel pipet. Use a calibrated pipet. Change pipet tip after every volume transfer. Reagents and assay components not equilibrated to room temperature prior to pipetting All reagents and assay components should be equilibrated to room temperature prior to pipetting.
Possible Causes Possible Solutions Low Bead Count Vacuum on for too long when aspirating buffer from wells Do not apply vacuum to the filter plate for longer than 10 sec after the buffer is completely drained from each well. Reader is clogged Refer to the troubleshooting guide in the Bio-Plex system hardware instruction manual (bulletin #10005042). Low Signal or Poor Sensitivity Standards reconstituted incorrectly Follow the standard preparation instructions carefully.
Possible Causes Possible Solutions Poor Recovery Expired Bio-Plex reagents were used Check that reagents have not expired. Use new or nonexpired components. Incorrect amounts of components were added Check your calculations and be careful to add the correct volumes. Microplate shaker set to an incorrect speed Check the microplate shaker speed and use the recommended setting. Setting the speed too high may cause splashing and contamination. Use the recommended plate shaker.
Possible Causes Possible Solutions Impact of Sample Matrix Negative MFI values in samples If samples contain little or no analyte, negative values observed may be due to statistical variation. If assay drift is suspected, retest the samples by positioning them next to the standards. If contamination of standards is suspected, check the standard replicate value and be careful when adding samples to the wells. Matrix effects could also produce negative sample values.
Plate Layout Template 32
Safety Considerations Eye protection and gloves are recommended when using these products. Consult the MSDS for additional information. The Bio-Plex Pro™ assays contain components of animal origin. This material should be handled as if capable of transmitting infectious agents. Use universal precautions. These components should be handled at Biosafety Level 2 containment (U.S. government publication: Biosafety in Microbiological and Biomedical Laboratories (CDC 1999)).
Ordering Information Detailed ordering information can be found at www.bio-rad.com/bio-plex. Catalog # Premixed 1 x 96-Well All-In-One Multiplex Kit Includes premixed magnetic capture beads, premixed detection antibodies, standards mix, 2-level controls, blocking buffer, standard diluent, lysate dilution buffer (LDB), cytosolic extraction buffer (CEB), 10x assay buffer, 10x streptavidin-PE, 96-well flat bottom plate, plate seals, and instructions.
Bio-Rad Laboratories, Inc. Life Science Group Web site www.bio-rad.
