Bio-Plex Pro Cell Signaling 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 Components and Storage 4 Recommended Materials 5 Assay Workflow 6 lmportant Considerations 7 Detailed Instructions 8 11. Prepare the Samples 22. Plan the Plate Layout 33. Prepare the Wash Method 44. Run the Assay 55.
Introduction Bio-Plex Pro™ Cell Signaling Assays Cell signaling is a complex process through which cells receive and respond to stimuli from the surrounding environment. For example, circulating cytokines and chemokines elicit a response from lymphocytes by binding to cell surface receptors and activating intracellular phosphoprotein signaling cascades.
Principle Technology The Bio-Plex® suspension array system is built upon the three core elements of xMAP technology: n n n luorescently dyed microspheres (also called beads), each with a distinct F color code or spectral address to permit discrimination of individual tests within a multiplex suspension.
Biomarker of Interest Streptavidin Magnetic Bead Biotinylated Detection Antibody Capture 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 Components and Storage Table 1. Components required for a complete 1 x 96-well cell signaling assay. Note that custom x-Plex™ assays include a premixed (multiplex) set of beads and detection antibodies in an all-in-one kit.
Table 2. Recommended materials. Item Ordering Information Bio-Plex Pro™ Assays Quick Guide 3 Bulletin #10024930 (download at www.bio-rad.
Assay Workflow Prewet wells (for filter plate only) Add 50 μl 1x beads to wells Wash 2x Add 50 μl samples, controls, and blanks; incubate overnight at RT with shaking Wash 3x Add 25 μl 1x detection antibody, incubate 30 min at RT with shaking Wash 3x Add 50 μl 1x streptavidin-PE, incubate 10 min at RT with shaking Wash 3x Add 125 μl of resuspension buffer, shake for 30 sec Acquire data 6
lmportant Considerations Assay Procedures n P lease pay close attention to vortexing, shaking, and incubation times and to Bio-Plex® reader PMT (RP1) setting, as these have been optimized specifically for the cell signaling assays n n n n F or optimal performance, use only reagents specific for Bio-Plex Pro™ cell signaling assays.
Detailed Instructions The following pages provide detailed instructions for each step of the assay procedure, including sample preparation, running the assay, and reading the plate with Bio-Plex Manager™ and Luminex xPonent software. 1.
Adherent Cells 1. Stop the treatment reaction by aspirating the culture medium and quickly rinsing the cells with ice-cold cell signaling cell wash buffer (bottle with the blue cap). The volume of buffer required is the same as the volume of aspirated cell culture medium. Keep the cells on ice during all steps when possible. 2. Completely remove the buffer before lysing the cells. 3. Immediately add the cell lysis buffer to the cells.
Suspension Cells 1. Collect cell suspension and pellet the cells by spinning at 1,000 x g for 5 min at 4°C. 2. Aspirate off cell culture medium completely. 3. Wash by resuspending the cells with ice-cold cell wash buffer (bottle with the blue cap). 4. Centrifuge the cells at 1,000 x g for 5 min at 4ºC. 5. Completely remove the buffer. 6. Immediately add the proper volume of cell lysis buffer and gently rock for 20 min at 4°C. 7.
Bio-Rad Cell Lysate Controls The positive and negative cell lysate controls are used for qualitative verification of assay performance. Refer to Table 3 to select the appropriate controls for your phosphoprotein or total target of interest. Table 3. Selection guide for Bio-Rad cell lysate controls.
Table 3. Selection guide for Bio-Rad cell lysate controls (continued).
1. Assign blank, negative control, positive control, and samples accordingly. Note: When designating blank using B , Bio-Plex Manager software will automatically subtract the blank MFI value from all other assay wells. 2. Once the total number of wells is known, see Tables 6–9 or the Calculation Worksheet on pages 32–33 to determine the required volumes of beads, detection antibodies, and streptavidin-PE to use.
3. Prepare the Wash Method The cell signaling assays are compatible with both magnetic separation and vacuum filtration methods. However, for best results, we recommend performing the assays in a flat bottom plate with magnetic separation. Table 4. Summary of compatible wash stations and plate types.
Table 5. Summary of wash steps and settings. After each assay incubation step, perform the appropriate wash step as shown below. Bio-Plex Pro Wash Station/Handheld Magnet/Vacuum Manifold* Wash Program Settings and Manual Wash Steps Assay Step # of Washes Volume, µl Magnetic Soak, min 1. Add beads to plate 2 200 1 2. Sample incubation 3 200 1 3. Detection Ab incubation 3 200 1 4. SA-PE incubation 3 200 1 * No magnetic soak for vacuum filtration.
4. Run the Assay Considerations n B ring all assay components and samples to room temperature before use n U se calibrated pipets and pipet carefully, avoiding bubbles n A ssay incubations are carried out in the dark. Cover the plate with aluminum foil or otherwise protect from extended exposure to light DAY 1 Prepare Samples and Controls 1. Thaw sample lysates and keep on ice (see Section 1, Prepare the Samples, for lysate preparation). 2.
7. Dilute coupled beads to 1x by pipetting the required volume into the tube containing wash buffer. Vortex. Each well of the assay requires 2.5 μl of the 20x stock adjusted to a final volume of 50 μl in wash buffer. Note: To minimize volume loss, use a 200–300 μl capacity pipet to remove beads from the 20x stock tube. If necessary, perform the volume transfer in 2 steps. Do not use a 1,000 μl capacity pipet and/or wide bore pipet tip. 8. Protect the beads from light with aluminum foil.
13. G ather the samples, Bio-Rad cell lysate controls, and blank. Use detection antibody diluent as the blank. Transfer 50 µl of each sample or blank to the appropriate wells of the plate. 14. Cover with a new sheet of sealing tape and incubate in the dark overnight (15–18 hr) at room temperature with shaking. Note: Fully resuspend the beads/sample mixture by vigorously shaking at 900–1,100 rpm for 30 sec. Slowly ramp up to speed to avoid splashing.
Preparing 1x detection antibodies from 20x stock (includes 25% excess volume): Table 8. Premixed panel or one singleplex assay. # of Wells 20x Detection Antibodies, µl Detection Antibody Diluent, µl 96 150 2,850 3,000 48 75 1,425 1,500 Total Volume, µl Table 9. Mixing two singleplex assays.
27. Add the required volume of detection antibody diluent to an appropriately sized polypropylene tube. This will be used to dilute SA-PE to 1x. 28. Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform a quick spin to collect the entire volume at the bottom of the vial. 29. Dilute SA-PE to 1x by pipetting the required volume into the tube containing detection antibody diluent. Vortex and protect from light until ready to use. Each well of the assay requires 0.
36. To resuspend beads for plate reading, add 125 µl resuspension buffer to each well. Cover the plate with a new sheet of sealing tape and shake at 900–1,100 rpm for 30 sec. 36. Slowly remove the sealing tape and place the plate on the reader to acquire data. Table 11. Read the plate using the appropriate instrument settings.
Start Up System (Bio-Plex 100, 200, or similar) 1. Empty the waste bottle and fill the sheath fluid bottle before starting if high throughput fluidics (HTF) are not present. This will prevent fluidic system backup and potential data loss. 2. Turn on the reader, XY platform, and HTF (if included). Allow the system to warm up for 30 min (if not already done). 3. Select Start up and follow the instructions. If the system is idle for 4 hr without acquiring data, the lasers will automatically turn off.
protocol. To create a new protocol, select File, then New from the main menu. Locate and follow the steps under Protocol Settings. 6. Click Describe Protocol and enter information about the assay (optional). 7. Click Select Analytes and create a new panel. Visually confirm the selected analytes and proceed to step 8. a. Click the Add Panel button in the Select Analytes toolbar. Enter a new panel name. Select Bio-Plex Pro Assay (Magnetic) or MagPlex Beads (Magnetic) from the assay dropdown list.
Table 12. Bead regions for cell signaling assays.
Fig. 3. Plate formatting. 10. Click Enter Sample Info and enter sample information and the appropriate dilution factor if any. 11. Click Run Protocol and confirm that the assay settings are correct. a. Refer to Table 11 for the recommended RP1 (PMT) setting. Protocols using alternative settings should be validated by the end user. b. Confirm that data acquisition is set to 50 beads per region.
Acquire Data 1. Shake the assay plate at 900–1,100 rpm for 30 sec, and visually inspect the plate to ensure that the assay wells are filled with buffer. Slowly remove the sealing tape before placing the plate on the plate carrier. 2. Click Run Protocol, and on the pop-up screen, select Load Plate and click OK to start acquiring data. 3. Use the Wash Between Plates command after every plate run to reduce the possibility of clogging the instrument. 4.
Luminex xPONENT Software 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. For the Bio-Plex MAGPIX reader, use the default instrument settings. 1.
Troubleshooting Guide This troubleshooting guide addresses problems that may be encountered with Bio-Plex Pro™ assays. If you experience any of the problems listed below, review the possible causes and solutions provided. Suboptimal assay performance may also be due to the Bio-Plex® suspension array reader. To eliminate this possibility, use the Bio-Plex validation kit to assist in determining if the array reader is functioning properly. Table 13. Troubleshooting guide.
Table 13. Troubleshooting guide (continued).
Table 13. Troubleshooting guide (continued).
Plate Layout Template 31
Calculation Worksheet If using either a premixed panel or one singleplex assay, follow these instructions. Plan the plate layout and enter the number of wells to be used in the assay:_______ 1 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl of coupled beads (1x): _______ x 50 µl = _______ µl 1 2 b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl 2 3 c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl 2 3 4 d.
If mixing two or more singleplex assays, follow these instructions. Enter the number of wells to be used in the assay:_______ 1 1. Determine the volume of 1x coupled beads needed. a. Each well requires 50 µl coupled beads (1x): _______ x 50 µl = _______ µl 1 2 b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl 2 3 c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl 2 3 4 d.
Safety Considerations Eye protection and gloves are recommended while using this product. Consult the MSDS for additional information. Human source material. Treat as potentially infectious. The lysates provided with Bio-Plex Pro™ cell signaling assays contain components of human origin. The components are known to contain an agent that requires handling at Biosafety Level 2 containment as defined by U.S.
Ordering Information Detailed ordering information can be found at www.bio-rad.com/bio-plex.
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