Bio-Plex Pro Human Chemokine 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 Wash Buffer 11 5. Prepare Standards and Controls 11 6. Prepare Samples 14 7. Prepare Coupled Beads 16 8. Run Assay 18 9.
Introduction Chemokines are small molecular weight (8–10 kD) cytokines secreted by various eukaryotic cell types, including those of the immune system. Their main function is to promote and regulate cell migration in both normal and pathological conditions, including immune surveillance, inflammation, angiogenesis, microbial infection, autoimmune diseases, tumor growth, vascular diseases, and transplant rejection (Locati et al. 2005, Slettenaar and Wilson 2006).
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 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 Contents and Storage Reagents Supplied Bio-Plex Pro™ human chemokine assays are available in a convenient all-in-one kit format that includes assay, reagent, and diluent components in a single box. Table 1. Contents of 1 x 96-well kits. Quantity*** Component Coupled magnetic beads (20x) 1 tube Detection antibodies (20x) 1 tube Standards 1 vial 1 vial Quality control* 1 bottle (8 ml) Sample diluent HB Detection antibody diluent HB 1 bottle (3.
Table 2. Recommended materials. Item Ordering Information Bio-Plex Pro Chemokine Assays Quick Guide Bulletin #10031991 (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, samples, controls; incubate on shaker at 850 rpm for 1 hr at RT Wash 3 x 100 μl Add 25 μl 1x detection antibody; incubate on shaker at 850 rpm for 30 min at RT Wash 3 x 100 μl Add 50 μl 1x streptavidin-PE; incubate on shaker at 850 rpm for 10 min at RT Wash 3 x 100 μl Resuspend in 125 μl assay buffer, shake at 850 rpm for 30 sec Acquire data on Bio-Plex system 6
lmportant Considerations Instruments and Software The Bio-Plex Pro™ 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 (see the Run Assay section).
1. Plan Plate Layout Determine the total number of wells in the experiment using the Plate Layout Template on page 35 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 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. Note: While the instrument is warming up, bring the 10x wash buffer, assay buffer, and diluents to room temperature. Keep other items on ice until needed. Also, begin to thaw frozen samples. Start up and calibrate the Bio-Plex system with Bio-Plex Manager™ software prior to setting up the assay.
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. 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 3.
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 to –1 to –3" Hg. In general, 100 µl liquid should take 3–4 sec to clear the well. For more detailed instructions, refer to bulletin #10005042. 4. Prepare Wash Buffer 1. Bring the 10x stock solution to room temperature. 2. If crystals exist, ensure that they are completely dissolved. Mix the 10x stock solution by inversion before preparing the 1x wash buffer.
Selecting a Diluent for Standards and Controls Refer to Table 4 for recommended diluents based on different sample types. In order to meet the lot-specific control ranges provided on the product data sheet, both the standards and controls should be reconstituted in Bio-Plex® standard diluent HB. If reconstituting in a different diluent, users will need to establish/validate their own control ranges or acceptance criteria. Table 4. Summary of recommended diluents for standards and controls.
4. During the incubation period, prepare the samples as instructed in the Prepare Samples section. Prepare the Standard Dilution Series The following procedure produces an eight-point standard curve with a fourfold dilution between each point. Pipet carefully using calibrated pipets and use a new pipet tip for every volume transfer. 1. Label eight 1.5 ml polypropylene tubes S2 through S8 and Blank.
6. Prepare Samples General guidelines for preparing different sample types are provided here. For more information, consult publications listed in Bio-Rad bulletin #5297, available for download at www.bio-rad.com, or contact Bio-Rad Technical Support. n 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 Prepare sample dilutions in microcentrifuge tubes.
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. Alternatively, filter the samples with a 0.8/0.2 μm dual filter to prevent clogging. 5. Dilute samples fourfold (1:4) by adding 1 volume of sample to 3 volumes of Bio-Plex® sample diluent HB (for example, 40 µl sample + 120 µl sample diluent HB). 6.
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.
3. Vortex the 20x stock of 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 requires 2.5 μl of the 20x stock adjusted to a final volume of 50 μl in assay buffer. 5.
8. 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 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 on a shaker at 850 ± 50 rpm at room temperature (RT). Cover the plate with sealing tape and protect from light with aluminum foil Table 8.
Add Coupled Beads, Samples, Standards, Blank, and Controls 1. Cover unused wells of the assay plate with sealing tape. 2. Prewet the filter plate. Skip this step if using a flat bottom plate. Prewet the wells with 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) beads for 30 sec at medium speed.
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 μl of the 20x stock adjusted to a final volume of 25 μl in detection antibody diluent. Preparing 1x detection antibodies from 20x stock (includes 25% excess volume) Table 9. 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 10.
2. Add the required volume of assay buffer to a 15 ml polypropylene tube. 3. Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform a 30 sec spin to collect the entire volume at the bottom of the tube. 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 of the assay requires 0.5 μl of the 100x stock adjusted to a final volume of 50 μl in assay buffer. Table 11.
Table 12. Read the plate using the appropriate instrument settings. 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 50 Bio-Plex® MAGPIX™ N/A, use default instrument settings * Or similar Luminex-based system. 9. 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.
a. Click Add Panel in the Select Analytes toolbar. Enter a new panel name. Select Bio-Plex Pro Assay Magnetic from the assay dropdown list. If using Bio-Plex Manager version 5.0 or lower, select MagPlex from the assay dropdown list. b. Click Add. Enter the bead region number and name for the first analyte. Click Add Continue to repeat for each analyte in the assay. Refer to the bead regions in parentheses ( ) listed on the peel-off label provided with the standards.
Note: Do not use preset panels found in Bio-Plex Manager software version 5.0 or earlier, as the bead regions are not up to date. 3. Click Format Plate and format the plate according to the plate layout created in section 1 (Plan Plate Layout). To modify the plate layout, follow the steps below (see Figure 4). a. Select the Plate Formatting tab. b. Select the standards icon S and drag the cursor over all the wells that contain 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. Optional: enter the lot number of the vial of standards into the Standard Lot box and click Save. 5. Click Enter Controls Info. a. For user-specified controls, select an analyte from the dropdown menu, then enter a description and concentration. Repeat for each additional analyte in the assay. b.
Acquire Data 1. 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 buffer. Slowly remove the sealing tape and any plate cover 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.
Data Analysis Quality Controls If the quality controls were run in the assay plate, open the results (.rbx) file, click on Report Table, and locate the control wells. Visually compare the observed concentrations of the high and low controls in the Report Table against the lot-specific control ranges shown 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 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 Pipet carefully when adding standards, controls, 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 Low Bead Count Miscalculation of bead dilution Possible Solutions 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 Assay plate not shaken enough during incubation steps and prior to reading 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. High Background Signal Poor Recovery Check that reagents have not expired.
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.
Appendix: Non-Human Primate (NHP) Cross-Reactivity The human chemokine assays were found to be cross-reactive with two common NHP species, cynomolgus (Cyno) macaque and rhesus macaque. Other NHP species were not tested. The degree of crossreactivity was determined based on the ability of each assay to detect native analyte in serum, plasma and/or culture media from mitogen stimulated peripheral blood mononuclear cells (PBMC).
Plate Layout Template 35
Calculation Worksheet If using either a premixed panel or one singleplex assay, follow these directions. 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 singleplex assays, follow these directions. 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 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 as defined by U.S. government publication, Biosafety in Microbiological and Biomedical Laboratories (Centers for Disease Control 1999).
Ordering Information Detailed ordering information can be found at www.bio-rad.com/bio-plex.
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