Bio-Plex Manager™ software recommended Bio-Plex Pro Assays Angiogenesis TM Instruction Manual For technical support, contact your local Bio-Rad office or in the US, call 1-800-4BIORAD (1-800-424-6723). For research use only. Not for diagnostic procedures.
Table of Contents Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Section 2 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Section 3 Product Description . . . . . . . . . . . . . . . . . . . . . . .4 Section 4 Required Materials . . . . . . . . . . . . . . . . . . . . . . . . 5 Section 5 Recommended Materials . . . . . . . . . . . . . . . . . . 6 Section 6 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . .
Section 1 Introduction Angiogenesis is a critical component of various normal and pathological processes. A regulated angiogenic process includes the growth of new blood vessels for tissue repair, fetal development and female reproductive cycle. An uncontrolled angiogenic growth contributes to a major pathological component of diseases such as cancer, cardiovascular disease, and rheumatoid arthritis.
Section 2 Principle Technology The Bio-Plex® suspension array system is built around three core technologies. The first is a novel technology that uses up to 100 unique fluorescently dyed beads (xMAP technology) that permit the simultaneous detection of up to 100 different types of molecules in a single well of a 96-well microplate. The second is a flow cytometer with two lasers and associated optics to measure the different molecules bound to the surface of the beads.
Assay Workflow Prewet wells Add beads Wash Add standards, controls, and samples, 30 min Wash Add detection antibody, 30 min Wash Add streptavidin-PE, 10 min Wash Resuspend, acquire data 3
Section 3 Bio-Plex® Reagent Kit Product Description Bio-Plex Pro™ angiogenesis assays require the use of the Bio-Plex® angiogenesis reagent kit to run the multiplex panel. Component of the Bio-Plex Angiogenesis Reagent Kit 171-304060 1x96-Well Format 171-304061 10x96-Well Format Bio-Plex assay buffer Store at 4ºC. Do not freeze. 1 x 75 ml 1 x 750 ml Bio-Plex wash buffer Store at 4ºC. Do not freeze. 1 x 150 ml 2 x 750 ml Bio-Plex detection antibody diluent Store at 4ºC. Do not freeze.
Section 4 Required Materials Bio-Plex Pro™ angiogenesis assays require the Bio-Plex Pro™ angiogenesis reagent kit and a multiplex angiogenesis assay panel. The Bio-Plex Pro angiogenesis assay panels contain the following components. • Antibody-conjugated beads (25x concentration) • Detection antibody (10x concentration) • Angiogenesis standard (2 vials, lyophilized) • Angiogenesis control (2 vials, lyophilized) Visit us on the web at www.bio-rad.com/bio-plex/ for our latest list of assays.
Section 5 Recommended Materials Item Bio-Plex human serum diluent kit For tissue culture samples, dilute samples and standards with appropriate tissue culture media Bio-Plex Pro human angiogenesis standards Additional standards sold separately (optional) Item Bio-Rad catalog #171-305000 (1x96) Bio-Rad catalog #171-305001 (10x96) Bio-Rad catalog #171-D40003 (2 vials, lyophilized) Bio-Rad catalog #171-D40004 (50 vials, lyophilized) Ordering Information Bio-Plex® suspension array system (or Luminex System)
Section 6 Sample Preparation This section provides instructions for preparing samples derived from serum, plasma, and tissue culture supernatant. For sample preparations not mentioned here, consult the publications listed in Bio-Rad bulletin 5297, available for download at discover.bio-rad.com Serum and Plasma Samples Note that for plasma samples, both EDTA plasma and citrate plasma are acceptable. Avoid using hemolyzed samples. 1.
Section 7 Standard Preparation Two vials of Iyophilized angiogenesis standards are provided in each Bio-Plex Pro™ angiogenesis assay. However, only one vial is required per 96-well plate. The product insert provided with the assay lists the concentration of the reconstituted standard. This procedure will prepare enough standard to run each dilution in duplicate. Reconstitute Standards 1.
3. Add 400 µl of reconstituted standard to the first 1.5 ml tube with 0 µl of standard diluent. This is identified as S1 in the diagram below and in the product insert provided with assay. 4. Continue making serial dilutions of the standard as shown. After making each dilution, vortex gently and change the pipet tip after every transfer. NOTE: Running an additional two 0 pg/ml blanks is strongly recommended. The blank wells are useful for troubleshooting and determining LOD.
Section 8 Control Preparation (Optional) Two vials of lyophilized angiogenesis control are provided in each Bio-Plex Pro™ Angiogenesis assay. The preparation of high, medium, and low controls is optional to monitor plate-to-plate variations. This section provides instructions on how to reconstitute the Iyophilized controls (refer to the product insert provided with the assay for reconstituted control values).
Example of Control Dilution Series 11
Section 9 Assay Instructions The following instructions apply to Bio-Plex Pro™ angiogenesis assays. All of the necessary components are provided premixed for ease of use. Plan Experiment 1. Assign which wells of a 96-well plate will be used for each standard, control, and sample (see the example below). 2. Determine the total number of wells that will be used in the assay.
Prepare Coupled Magnetic Beads Protect the beads from light by covering the tubes with aluminum foil. Keep all tubes on ice until ready to use. 1. Vortex the coupled beads (25x) at medium speed for 15-20 sec. 2. Prepare a sufficient volume of coupled beads (1x) using assay buffer. Each well requires 2 µl of coupled beads (25x) adjusted to a final volume of 50 µl with assay buffer (refer to the example below).
Assay Procedure Bring all buffers to room temperature. Avoid bubbles when pipetting. Assay Key – The following terms are repeated throughout the assay procedure. Refer to these detailed instructions when wash, incubate, and vacuum-filter are shown in bold. Add 100µL of wash buffer to each well. Place the filter plate on a calibrated vacuum apparatus and remove the buffer by vacuum filtration. Blot the bottom of the filter plate with a clean paper towel. Repeat as specified.
7. Prepare a sufficient volume of detection antibodies (1x) using detection antibody diluent. Each well requires 2.5 µl of detection antibodies (10x) adjusted to a final volume of 25 µl with detection antibody diluent (refer to the example below). Example Detection Antibody Calculations # of Wells 10x Detection Antibody (µl) Detection Antibody Diluent (µl) Total Volume (µl) 96 300 2,700 3,000 48 150 1,350 1,500 32 100 900 1,000 24 75 675 750 8.
Example Streptavidin-PE Calculations 100x Assay Buffer (µl) Streptavidin-PE # of Wells (µl) Total Volume (µl) 96 60 5,940 6,000 48 30 2,970 3,000 32 20 1,980 2,000 24 15 1,485 1,500 13. After the detection antibody incubation, slowly remove and discard the sealing tape, then vacuum-filter. 14. Wash 3 times. 15. Vortex the streptavidin-PE (1x) vigorously and add 50 µl to each well. Incubate for 10 min. 16.
Section 10 Data Acquisition Bio-Plex Manager™ software is recommended for Bio-Plex Pro™ angiogenesis assays. Refer to the details in the appropriate section below for your xMAP system software package. For instructions using other xMAP system software packages, contact Bio-Rad technical support or your Bio-Rad field applications specialist. NOTE: To minimize protocol setup, lot-specific Bio-Plex Pro angiogenesis assay protocols for Bio-Plex Manager version 4.0 and higher are available for download at www.
1. Select Calibrate and confirm that the default values for CAL1 and CAL2 are the same as the values on the Bio-Plex calibration bead labels. Use the Bio-Plex low RP1 target value. 2. Select OK and follow the instructions for CAL1 and CAL2 calibration. Bio-Plex Manager Software version 4.0, 4.1 and 4.1.1 This software versions permit calibration at either low or high RP1 target values. The selection of which target value to use is assay dependent.
a) Click the Add Panel button in the Select Analytes toolbar. b) Enter a name for the new panel in the top field. Select the “BioPlex Pro” assay type from the pull down menu (only for version 5.0 or higher). Then click Add to add analytes. c) Enter the bead region number of the first analyte in the Region field, and the analyte name in the Name field. NOTE: The bead region number must be correct for proper detection of analytes.
Plate Formatting Example 6. 20 Select Step 4 (Enter Standards Info) to enter standards information. Skip to the next step if you are using a downloaded protocol from the website; this information will already be entered. a) Deselect the box labeled “same concentration values for all analytes”. b) Select the first analyte from the pull-down cell. c) Click on the Enter Automatically button and then select the most concentrated value (typically S1). d) Enter the value for the highest concentration.
7. Select Step 5 (Enter Controls Info) to enter controls information. This is where the concentration of the user-specified controls is entered into the protocol. a) If necessary, deselect the box for same concentration values for all analytes. b) Select an analyte from the pull down cell. c) Enter the description, concentration, and dilution information for each user-specified control. d) Repeat steps 7b and 7c for each additional analyte in the assay. 8.
d) In Advanced Settings, confirm that the default DD gate values are set to 5000 (low) and 32000 (high). e) Select Start, save the .rbx file, and begin data acquisition. Bio-Plex Manager Software version 4.0 Note that this version does not include the 25 Bead Map. Hence, the 100 bead map is used. 5. a) Specify data acquisition for 100 beads per region. b) In Advanced Settings, set the sample size to 50 µl.
Reacquire Data It is possible to aquire data from a well or plate a second time using the Rerun/Recovery mode located below Start in Step 7 (Run Protocol). 1. Check the wells where data will be acquired a second time. Any previous data will be overwritten. 2. Remove the buffer by vacuum filtration and add 100 µl of assay buffer to each well. Cover the filter plate with a new sheet of sealing tape. 3. Repeat Acquire Data steps 1-6 to acquire data a second time.
Section 11 Troubleshooting Guides This troubleshooting guide addresses problems that may be encountered with Bio-Plex Pro™ angiogenesis assays. If you experience any of the problems listed below, review the possible causes and solutions provided. This will assist you in resolving problems directly related to how the assay steps should be performed. Poor assay performance may also be due to the Bio-Plex® suspension array reader.
Possible Causes Possible Solutions Pipetting technique Pipet carefully and slowly 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 were not equilibrated to room temperature prior to plating All reagents and assay components should be equilibrated to room temperature prior to plating.
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 15–20 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 High Background Signal Incorrect buffer was used (for example, assay buffer used to dilute standards) Use sample matrix or serum standard diluent to dilute the standards. Spiked “0 pg/ml” wells by mistake Be careful when spiking standards. Do not add any antigens in the 0 pg/ml (blank) point. Streptavidin-PE incubated too long Follow the procedure incubation time. Poor Recovery Expired Bio-Plex reagents were used Check that reagents have not expired.
Section 12 Safety Considerations Eye protection and gloves are recommended while using this product. Consult the MSDS for additional information. The Bio-Plex Pro™ angiogenesis assays contain components of animal origin. This material should be handled as if capable of transmitting infectious agents. Please use universal precautions. These components should be handled at Biosafety Level 2 containment [US Government publication: Biosafety in Microbiological and Biomedical Laboratories (CDC, 1999)].
xMAP is a trademark of Luminex Corp. Costar is a trademark of Coming Costar Corporation. Eppendorf is a trademark of Eppendorf-Netheler-Hinz GmbH. Luminex 100, xPONENET, and xMAP are trademarks of Luminex Corporation. Multiscreen is a trademark of Millipore Corporation. Vortex-Genie is a trademark of Scientific Industries, Inc.
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