Bio-Rad Bio-Plex Pro Human Angiogenesis Reagent and Diluent Kits User Manual

Bio-Plex

Manager™ software

recommended

Bio Plex ProTMAssays

Angiogenesis

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 . . . . . . . . . . . . . . . . . . . . . . . 7

Section 7 Standard Preparation . . . . . . . . . . . . . . . . . . . . . 8

Section 8 Control Preparation (optional) . . . . . . . . . . . . . 10

Section 9 Assay Instructions . . . . . . . . . . . . . . . . . . . . . . . 12

Plan Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Prepare Coupled Magnetic Beads . . . . . . . . . . . . 13

Calibrate Vacuum Apparatus . . . . . . . . . . . . . . . . 13

Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Section 10 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . 17

Section 11 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . 24

Section 12 Safety Considerations . . . . . . . . . . . . . . . . . . . .28

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. Many proteins involved in the uncontrolled angiogenic growth are candidate drug targets relevant for the development of cancer therapies. Developing these therapies involves measurement of various angiogenesis biomarkers.

Bio-Plex Pro™ angiogenesis assays are magnetic bead-based multiplex assays that allow the measurement of angiogenesis biomarkers in diverse matrices including serum, plasma, and cell/tissue culture supernatant. The multiplexing feature makes it possible to quantitate the level of multiple angiogenesis targets in a single well of a 96-well microplate in just 3 hours, using as little as 12.5 µl of serum or plasma, or 50 µl of culture supernatant.

As one of the most recent additions to the Bio-Plex system, these assays incorporate magnetic beads into their design. The magnetic beads allow the use of an assay protocol similar to non-magnetic Bio-Plex assays, with the option of using magnetic separation of wash steps instead of vacuum filtration (and allows automation of many of the steps).

suspension array

Bio-Plex Manager™ software is recommended for Bio-Plex Pro angiogenesis assays. Instructions for Luminex xPONENT software are also provided. For instructions using other xMAP system software packages, contact Bio-Rad Technical support or your Bio-Rad field application specialist.

For a current listing of Bio-Plex angiogenesis assays, visit us on the Web at

www.bio-rad.com/bio-plex/

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. The third is a high-speed digital signal processor that efficiently manages the fluorescent output.

Assay Format

The principle of these 96-well plate-formatted, bead-based assays is similar to a capture sandwich immunoassay. An antibody directed against the desired angiogenesis target is covalently coupled to internally dyed beads. The coupled beads are allowed to react with a sample containing the angiogenesis target. After a series of washes to remove unbound protein, a biotinylated detection antibody specific for a different epitope is added to the reaction. The result is the formation of a sandwich of antibodies around the angiogenesis target. Streptavidinphycoerythrin (streptavidin-PE) is then added to bind to the biotinylated detection antibodies on the bead surface.

Data Acquisition and Analysis

Data from the reaction are then acquired using the Bio-Plex suspension array system (or Luminex system), a dual-laser, flow-based microplate reader system. The contents of the well are drawn up into the reader. The lasers and associated optics detect the internal fluorescence of the individual dyed beads as well as the fluorescent signal on the bead surface. This identifies each assay and reports the level of target protein in the well. Intensity of fluorescence detected on the beads indicates the relative quantity of targeted molecules. A high-speed digital processor efficiently manages the data output, which is further analyzed and presented as fluorescence intensity on Bio-Plex Manager™ software.

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

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

Bio-Plex assay buffer Store at 4ºC. Do not freeze.

Bio-Plex wash buffer Store at 4ºC. Do not freeze.

Bio-Plex detection antibody diluent Store at 4ºC. Do not freeze.

Streptavidin-PE (100x) Store at 4ºC. Do not freeze.

Sterile filter plate (96-well) with cover and tray

Sealing tape 1 pack of 4 10 packs of 4 (40)

Angiogenesis Instruction Manual

Storage and Stability

Kit components should be stored at 4ºC and never frozen. Coupled magnetic beads and streptavidin-PE should be stored in the dark. All components are guaranteed for up to 6 months from the date of purchase when stored as specified in this manual.

171-304060

1x96-Well Format

1 x 75 ml 1 x 750 ml

1 x 150 ml 2 x 750 ml

1 x 15 ml 1 x 150 ml

1 vial 1 vial

1 plate 10 plates

1 1

10x96-Well Format

171-304061

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 Ordering Information

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 Ordering Information

Bio-Plex®suspension array system (or Luminex System)

Bio-Plex validation kit

Bio-Plex calibration kit

Microtiter plate shaker

IKA-Schuttler MTS-4 shaker for 4 microplates or Lab-Line Model 4625 Plate Shaker (or equivalent, capable of 300-1,100 rpm)

Filter plate vacuum apparatus

Bio-Rad Aurum™ vacuum manifold IMPORTANT: The use of filter plate manifolds other than the one specified may result in diminished assay performance; see section 8 for instructions specific to this assay

Vortexer

VWR brand mini-vortexer Scientific Instruments Vortex-Genie 2 mixer

Reagent reservoir

Corning, Inc. Costar 50 ml reagent reservoir 4870

Other materials

Pipets and pipet tips, sterile distilled water, aluminum foil, absorbent paper towels, 1.5 ml microcentrifuge tubes, 15 ml culture tubes

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)

Bio-Rad catalog #171-000205

Bio-Rad catalog #171-203001

Bio-Rad catalog #171-203060

IKA catalog #3208000

VWR catalog #57019-600

Bio-Rad catalog #732-6470

VWR catalog #58816-121

VWR catalog #58815-234

Bio-Rad catalog #224-4872

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. Collect and process the serum or plasma samples and assay immediately or freeze at –20ºC. Avoid repeated freeze-thaw cycles.

2. Centrifuge the samples at 13,200 rpm for 10 min at 4ºC to clear the samples of precipitate. Alternatively, carefully filter the samples with a

0.8/0.22 µm dual filter to prevent instrument clogging.

3. Immediately dilute 1 volume of sample with 3 volumes of sample diluent. Keep the samples on ice until ready for use.

Tissue Culture Supernatant Samples

1. Collect and process the tissue culture supernatant samples and assay immediately or freeze at –20ºC. Avoid repeated freeze thaw cycles.

2. If required, dilute the culture supernatant with culture medium. Serum-free culture medium should contain carrier protein (such as BSA) at a concentration of at least 0.5%. Keep the samples on ice until ready for use.

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. Gently tap the glass vial containing the lyophilized angiogenesis standard on a solid surface to ensure the pellet is at the bottom.

2. Reconstitute 1 vial of lyophilized standard with 500 µl of the standard diluent. Do not use assay buffer to dilute standards.

3. Gently vortex 1–3 sec and incubate on ice for 30 min.

4. Be consistent with the incubation time for optimal assay performance.

Prepare Standard Dilution Series

The angiogenesis concentrations specified for the 8-point standard dilution set have been selected for optimized curve fitting using the 5parameter logistic (5PL) or 4-parameter logistic (4PL) regression in Bio-Plex Manager™ software. Results generated using dilution points other than those listed in this manual have not been optimized.

1. Label a set of 1.5 ml Eppendorf tubes as shown in the diagram on the next page.

2. Pipet the appropriate volume of standard diluent into the tubes (see diagram on next page). Use serum standard diluent for serum and plasma samples and culture medium for culture samples.

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