Bio-Plex Pro Cytokine, Chemokine, and Growth Factor 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 Important Considerations 7 Detailed Instructions 1. Plan Plate Layout 8 2. Prepare Instrument 9 3. Prepare Wash Method 10 4. Prepare Standards 11 5. Prepare Samples 16 6. Prepare Coupled Beads 19 7. Run Assay 22 8.
Introduction Cytokines, chemokines, and growth factors are a diverse group of cell signaling proteins expressed and secreted by virtually all cell types, including cells of endothelial, epithelial, and immune origin. These proteins interact with specific receptors on target cells to mediate important physiological responses such as growth, immunity, inflammation, and hematopoiesis.
Principle Technology The Bio-Plex® multiplex system is built upon the three core elements of xMAP technology: n n n Fluorescently dyed 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 Reagents Supplied Bio-Plex Pro™ cytokine assays are offered in a convenient kit format that includes assay, reagent, and diluent components in a single box (Table 1). Table 1. Contents of Bio-Plex Pro cytokine, chemokine, and growth factor assays.* 1 x 96-Well 10 x 96-Well Component Format Format Standard diluent* 10 ml Sample diluent* 40 ml 80 ml Assay buffer 50 ml 500 ml Wash buffer 200 ml 1.
Table 2. Recommended materials. Item Ordering Information Bio-Plex Pro Assays Quick Guide 4 Bulletin #10024985 (download at www.bio-rad.
Assay Workflow Prewet wells (for filter plate only) Add 50 μl 1x beads to wells Wash 2 x 100 μl Add 50 μl standards, blank, samples, incubate at RT with shaking at 850 rpm (incubation time varies by assay) Wash 3 x 100 μl Add 25 μl 1x detection antibody, incubate 30 min at RT with shaking at 850 rpm Wash 3 x 100 μl Add 50 μl 1x streptavidin-PE, incubate 10 min at RT with shaking at 850 rpm Wash 3 x 100 μl Resuspend in 125 μl assay buffer, shake at 850 rpm for 30 sec Read plate on Bio-Plex system
Important Considerations Instruments and Software The cytokine 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 Prior to running the assay, determine the total number of wells in the experiment using the Plate Layout Template on page 40 or the Plate Formatting tab in Bio-Plex Manager™. 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. The blank should consist of your chosen standard diluent.
2. Prepare Instrument Start up and calibrate the Bio-Plex® 100/200 or similar 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. To prepare either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader, consult its respective user manual. The validation kit should be run monthly to ensure performance of fluidics and optics systems.
3. Prepare Wash Method Bio-Plex Pro™ 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.
4. Prepare Standards General Instructions n n n It is essential to reconstitute and dilute standards exactly as described in this section. Incorrect preparation may lead to low signal, high background, or inconsistent measurements from plate to plate The peel-off label provided with the standards lists the concentration of the most concentrated dilution point, S1.
RP1 (PMT) Setting for Standard Curves The Bio-Plex 200 and 3D systems have two RP1 (PMT or photomultiplier tube) setting options, while the Bio-Plex® MAGPIX™ has no PMT and therefore no PMT setting options. Instead, MAGPIX uses default instrument settings similar to low PMT on the Bio-Plex 200 (Table 6). Table 6. Overview of PMT setting options on Bio-Plex systems.
2. Add 500 μl of the appropriate diluent (see Table 5). Do not use assay buffer to reconstitute the standards. 3. Gently vortex the reconstituted standard for 5 sec then incubate on ice for 30 min. Be consistent with the incubation time in every assay to ensure best results. 4. During the incubation period, prepare the samples as instructed in the Prepare Samples section.
Reconstituting Standards for Cross-Panel Plexing Follow these directions when mixing human or mouse cytokine and diabetes assays. Note that rat diabetes and cytokine standards are premixed into one standard vial. Therefore, no extra mixing is required. Two mixing scenarios are provided in Figures 4 and 5 for detection at high and low PMT respectively.
4. Add 150 μl of standard diluent to the remaining tubes, as shown in Figure 4. 50 12.8 µl 500 µl Reconstituted Cytokine Standard 50 50 50 50 50 Transfer Volume (µl) 50 128 µl 500 µl Reconstituted Diabetes Standard 59.2 µl Standard Diluent “S1” Tube, 200 µl Total 150 150 150 150 150 150 150 150 S2 S3 S4 S5 S6 S7 S8 Blank Diluent (µl) Fig. 4. S1 mixture and fourfold dilution series of diabetes and cytokine standards for detection at high PMT.
5. Prepare Samples General guidelines for preparing different sample types are provided here. For more information, contact Bio-Rad Technical Support. n n Once thawed, keep samples on ice. Prepare dilutions just prior to the start of the assay and equilibrate to room temperature before use Do not freeze diluted samples Table 8. Summary of recommended sample diluents and dilution factors.
2. For serum, allow blood to clot at room temperature for 30 to 45 min. For plasma, proceed directly to the centrifugation steps. 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 platelets and precipitates, centrifuge again at 10,000 x g for 10 min at 4°C. 5.
Lysates The Bio-Plex cell lysis kit is required for lysate preparation (available separately, catalog #171-304011 and #171-304012). Refer to bulletin #5297 for a list of published articles on cytokine analysis in tissue samples. 1. Prepare the cell or tissue lysates according to the instructions provided with the Bio-Plex cell lysis kit. The protease inhibitors factor I and factor II are included in the kit. PMSF needs to be added to lysis buffer at a final concentration of 2 mM.
For the mouse ICAM-1 assay, dilute serum or plasma 1:200 in Bio-Plex serum-based diluent, which is included in the Bio-Plex Pro high dilution reagent kit, catalog #171-304080M. For example: n First dilution (1:10)—10 µl sample + 90 µl diluent n Second dilution (1:20)—10 µl from the first dilution + 190 µl diluent 6. Prepare Coupled Beads Instructions are provided for diluting the coupled beads to a 1x concentration.
3. Vortex the stock coupled beads at medium speed for 30 sec. Carefully open the cap and pipet any liquid trapped in the cap back into the tube. This is important to ensure maximum bead recovery. Do not centrifuge the vial; doing so will cause the beads to pellet. 4. Dilute coupled beads to 1x by pipetting the required volume into the 15 ml tube. Vortex. Each well of the assay plate requires either 2.5 µl (20x stock) or 5.0 µl (10x stock) adjusted to a final volume of 50 µl in assay buffer. 5.
Preparing 1x coupled beads from 20x stock (includes 20% excess volume) Table 12. Premixed panel or one singleplex assay. # of Wells 20x Beads, µl Assay Buffer, µl Total Volume, µl 96 288 5,472 5,760 48 144 2,736 2,880 Table 13. Mixing two singleplex assays or a premixed panel + singleplex assay. # of Wells 20x Beads, µl Singleplex #1 20x Beads, µl Singleplex #2 Assay Buffer, µl Total Volume, µl 96 288 288 5,184 5,760 48 144 144 2,592 2,880 Table 14.
7. Run Assay Considerations n n n n Bring all assay components and samples to room temperature before use Use calibrated pipets and pipet carefully, avoiding bubbles. Use new pipet tips for every volume transfer Pay close attention to vortexing, shaking, and incubation instructions. Deviation from the protocol may result in low assay signal and assay variability Assay incubations are carried out in the dark on a shaker at 850 ± 50 rpm.
Add Coupled Beads, Standards, and Samples 1. Cover unused wells with sealing tape. 2. Prewet the filter plate. Skip this step if using a flat-bottom plate. Prewet the wells using 100 µl assay buffer and remove the liquid by vacuum filtration. Dry the bottom of the filter plate thoroughly by blotting on a clean paper towel. 3. Vortex the diluted (1x) coupled beads for 30 sec at medium speed. Pour the diluted coupled beads into a reagent reservoir and transfer 50 µl to each well of the assay plate.
Prepare and Add Detection Antibodies Instructions are provided for diluting the detection antibodies to a 1x concentration. When mixing diabetes and cytokine assays, keep in mind the stock concentrations of detection antibodies as shown below. Table 17. Stock concentration of detection antibodies. Stock Concentration of Detection Antibodies Assay Human and mouse cytokines (groups I, II) 10x Mouse cytokines (group III) 20x Rat cytokines (group I) 20x Human, mouse, and rat diabetes 20x 1.
Table 19. Mixing two singleplex assays or a premixed panel + singleplex assay. # of Wells 10x Detection Antibodies, µl Singleplex #1 10x Detection Antibodies, µl Singleplex #2 Detection Antibody Diluent, µl Total Volume, µl 96 300 300 2,400 3,000 48 150 150 1,200 1,500 Preparing 1x detection antibodies from 20x stock (includes 25% excess volume) Table 20. Premixed panel or one singleplex assay.
8. over plate with a new sheet of sealing tape and protect from light C with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 30 min at room temperature. Prepare and Add Streptavidin-PE (SA-PE) 1. While the detection antibodies are incubating, use Table 23 or the Calculation Worksheet on page 41 to calculate the volume of SA-PE (100x) and assay buffer needed. Streptavidin-PE should be prepared 10 min before use. 2. Add the required volume of assay buffer to a 15 ml polypropylene tube. 3.
8. Cover plate with a new sheet of sealing tape and protect from light with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 10 min at room temperature. 9. After the streptavidin-PE incubation step, slowly remove and discard the sealing tape. 10. Wash the plate three times with 100 µl wash buffer. 11. To resuspend beads for plate reading, add 125 µl of assay buffer to each well. Cover the plate with a new sheet of sealing tape.
8. Read Plate Bio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay data acquisition and analysis. Instructions for Luminex xPONENT software are also included. For instructions using other xMAP system software packages, contact Bio-Rad Technical Support or your regional Bio-Rad field applications specialist. Prepare Protocol in Bio-Plex Manager Software v 6.0 and Higher The protocol should be prepared in advance so that the plate is read as soon as the experiment is complete.
2. Click Select Analytes and create a new panel. Visually confirm the selected analytes and proceed to step 3. a. Click the Add Panel button in the Select Analytes toolbar. Enter a new panel name. Select Bio-Plex Pro Assay Magnetic from the assay pull-down menu. If using Bio-Plex Manager version 5.0 or lower, select MagPlex from the assay pull-down menu. b. Click the Add button. Enter the bead region number and name for the first analyte.
Table 25. Bead regions for Bio-Plex Pro cytokine assays.
Fig. 6. Plate formatting. 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 included on the peel-off sticker provided with each vial of standards. b. Enter a dilution factor of 4 and click Calculate. The concentrations for each standard point will be populated for all analytes in the table. c.
7. Click Run Protocol and confirm that the assay settings are correct. a. Refer to Table 24 for the recommended RP1 (PMT) setting. Protocols using alternative PMT settings should be validated by the end user, for example when mixing diabetes assays with cytokine assays. b. Confirm data acquisition is set to 50 beads per region.
Reacquire Data It is possible to acquire data from a well or plate a second time using the Rerun/Recovery mode located below Start in the Run Protocol step. Any previous data will be overwritten. 1. Check the wells from which data will be reacquired. 2. Remove the buffer with the wash method of choice. 3. Add 100 µl assay buffer to each well. Cover the filter plate with a new sheet of sealing tape. Shake the plate at 850 ± 50 rpm for 30 sec.
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. Poor assay performance may also be due to the Bio-Plex® suspension array reader. To eliminate this possibility, use the validation kit to assist in determining if the array reader is functioning properly.
Possible Causes Possible Solutions High Intra-Assay CV Improper pipetting technique Reagents and assay components not equilibrated to room temperature prior to pipetting Contamination with wash buffer during wash steps 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.
Possible Causes Possible Solutions Low Bead Count Miscalculation of bead dilution Check your calculations and be careful to add the correct volumes. Beads clumped in multiplex bead stock tube Vortex for 30 sec at medium speed before aliquoting beads. 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.
Possible Causes Possible Solutions Incorrect buffer was used (for example, assay buffer used to dilute standards) Use standard diluent or diluent similar to final sample matrix to dilute standards. Accidentally spiked blank wells Do not add any antigens to the blank wells. Detection antibodies or streptavidin-PE incubated too long Follow the procedure incubation time precisely.
Possible Causes Possible Solutions Poor Recovery 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. Impact of Sample Matrix Negative MFI values in samples or standards If samples contain little or no analyte, negative values observed may be due to statistical variation.
Plate Layout Template 40
Calculation Worksheet If using either a premixed panel or one singleplex assay with 20x stocks of beads and detection antibodies, follow these directions. If using 10x stocks, divide by 10 instead of 20 in steps 1d and 2d. . 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.
If mixing singleplex assays with 20x stocks of beads and detection antibodies, follow these directions. If using 10x stocks, divide by 10 instead of 20 in steps 1e and 2e. 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.
If mixing diabetes assays (20x bead and detection antibody stocks) with cytokine assays (10x stocks), follow these directions. Note: Refer to Table 26 for the maximum number of diabetes and cytokine assays that may be multiplexed. Mixing across panels is not applicable to NHP diabetes assays. Table 26. Maximum number of singleplex diabetes and cytokine analytes that may be multiplexed.
2. Determine the volume of 1x diabetes and cytokine detection antibodies needed. a) Each well requires 25 μl of detection antibodies (1x): _______ x 25 μl = ______ μl 1 13 b) Include 25% excess to ensure enough volume: _______ μl x 0.
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.
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