Bio-Rad Bio-Plex Pro Human Chemokine Assays User Manual

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. Read Plate 22

Troubleshooting Guide 29

Appendix: Non-Human Primate (NHP) Cross-Reactivity 34

Plate Layout Template 35

Calculation Worksheet 36

Safety Considerations 38

Legal Notices 38

Ordering Information 39

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). The regulatory functions of chemokines are exerted
via binding and signaling through specific G protein–coupled receptors
expressed on the surface of chemokine-responsive cells.

Chemokines are classified into four subfamilies (C, CC, CXC, and CX3C)
based on the number and spacing of cysteine residues within the protein
sequence. The C chemokines are known as lymphotactins, and are
found at high levels in spleen, thymus, intestine, and peripheral blood
leukocytes. The CC chemokines have the first two cysteines in adjacent
positions and are known to attract granulocytes and lymphocytes,
including NK cells. The CXC chemokines have the first two of four
cysteines separated by a single amino acid, denoted X. Most CXC
chemokines are chemo-attractants for neutrophils and lymphocytes. The
CX3C chemokines have three amino acids inserted between the first two
cysteines. The only CX3C chemokine discovered to date is fractalkine,
which is both a chemo-attractant and adhesion molecule.

Multiplexing with Bio-Plex Pro Chemokine Assays

Bio-Plex Pro chemokine assays enable researchers to quantify multiple
protein biomarkers in a single well of a 96-well plate in just 3–4 hours.
These robust immunoassays require as little as 12.5 l of serum or
plasma or 50 l of other biological fluid. The use of magnetic (MagPlex)
beads allows researchers to automate wash steps on a Bio-Plex Pro
(or similar) wash station. Magnetic separation offers greater convenience
and reproducibility compared to vacuum filtration.

For more information please visit www.bio-rad.com/bio-plex.

1

Principle

Technology

The Bio-Plex® multiplex system is built upon the three core elements of
xMAP technology:

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. This allows simultaneous
detection of up to 500 different molecules in a single well of a 96-well
microplate on the Bio-Plex
on the Bio-Plex
Bio-Plex

n

A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems

®

®

200 system, and up to 50 different molecules on the

MAGPIX™ system

are flow cytometry–based instruments with two lasers and associated
optics to measure the different molecules bound to the surface of the
beads. In the Bio-Plex MAGPIX system, the sample is injected into a
chamber where the beads are imaged using LED and CCD technology

n

A high-speed digital signal processor that efficiently manages the

fluorescence data

Assay Format

Bio-Plex Pro™ assays are essentially immunoassays formatted on
magnetic beads. The assay principle is similar to that of a sandwich
ELISA (Figure 1). Capture antibodies directed against the desired
biomarker are covalently coupled to the beads. Coupled beads react
with the sample containing the biomarker of interest. After a series of
washes to remove unbound protein, a biotinylated detection antibody
is added to create a sandwich complex. The final detection complex is
formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate.
Phycoerythrin serves as a fluorescent indicator, or reporter.

®

3D system, up to 100 different molecules

2

Biomarker
of Interest

Streptavidin

Magnetic Bead

Capture

Antibody

Fig. 1. Bio-Plex sandwich immunoassay.

Biotinylated

Detection

Antibody

Phycoerythrin

Fluorescent

Reporter

Data Acquisition and Analysis

Data from the reactions are acquired using a Bio-Plex system or similar
Luminex-based reader. When a multiplex assay suspension is drawn into
the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates
the fluorescent dyes within each bead to provide bead classification and
thus assay identification. At the same time, a green (532 nm) laser excites
PE to generate a reporter signal, which is detected by a photomultiplier
tube (PMT). A high-speed digital processor manages data output, and
Bio-Plex Manager
intensity (MFI) as well as concentration (pg/ml). The concentration of
analyte bound to each bead is proportional to the MFI of reporter signal.

Using Bio-Plex Data Pro
can be combined into a single project for easy data management, quick
visualization of results, and simple statistical analysis.

™

software presents data as median fluorescence

™

software, data from multiple instrument runs

3

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.

Component

Coupled magnetic beads (20x)
Detection antibodies (20x)
Standards
Quality control*
Sample diluent HB
Detection antibody diluent HB
Standard diluent HB
Assay buffer
Wash buffer (10x)
Streptavidin-PE (100x)
Assay plate (96-well flat bottom plate)**
Sealing tape
Assay quick guide
Product data sheet

* Provided with the 40-plex fixed panel only.
** Filter plate option available with custom x-Plex
*** Volumes shown are approximate.

Quantity

1 bottle (8 ml)

1 bottle (3.5 ml)

1 bottle (10 ml)
1 bottle (50 ml)
1 bottle (60 ml)

1 pack of 4

™

and Express kits.

***

1 tube
1 tube

1 vial
1 vial

1 tube
1 plate

1 booklet

1 sheet

Storage and Stability

Kit contents should be stored at 4°C and never frozen. Coupled magnetic
beads and streptavidin-PE should be stored in the dark. All components
are guaranteed for a minimum of six months from the date of purchase
when stored as specified.

4

Table 2. Recommended materials.

Item

Bio-Plex Pro Chemokine Assays Quick Guide

Ordering Information

Bulletin #10031991 (download
at www.bio-rad.com/bio-plex)

®

Bio-Plex
Bio-Plex validation kit

200 system or Luminex system with HTF

Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001

Note: Run the validation kit monthly to ensure optimal

performance of fluidics and optics systems

Bio-Plex calibration kit

Bio-Rad catalog #171-203060

Note: Run the calibration kit daily to standardize

fluorescence signal

Bio-Plex Pro wash station

Bio-Rad catalog #300-34376

For use with magnetic bead-based assays only

Bio-Plex Pro II wash station

Bio-Rad catalog #300-34377
For use with both nonmagnetic and magnetic
bead-based assays

Bio-Plex handheld magnetic washer

Bio-Rad catalog #170-20100
For use with magnetic bead–based assays only

Bio-Plex Pro flat bottom plates (40 x 96-well)

Bio-Rad catalog #171-025001
For magnetic separation on the Bio-Plex Pro wash station

Titertube

®

micro test tubes

Bio-Rad catalog #223-9390
For preparing replicate standards, samples, and controls
prior to loading the plate

Microtiter plate shaker

IKA MTS 2/4 shaker for 2 or 4 microplates
or

Barnstead/Lab-Line Model 4625 plate

IKA catalog #320-8000

VWR catalog #57019-600
shaker (or equivalent capable of 300–1,100 rpm)

™

vacuum manifold

Aurum

Bio-Rad catalog #732-6470
For vacuum filtration

BR-2000 vortexer
Reagent reservoirs, 25 ml

For capture beads and detection antibodies

Reagent reservoir, 50 ml (for reagents and buffers)
Pall Life Science Acrodisc: 25 mm PF syringe filter

(0.8/0.2 µm Supor membrane)

Filter plate, 1 x 96 with clear plastic lid and tray

Bio-Rad catalog #166-0610

VistaLab catalog #3054-1002

or

VistaLab catalog #3054-1004

VistaLab catalog #3054-1006

Pall Life Sciences

catalog #4187

Bio-Rad catalog #171-304502

Other: 15 ml polypropylene tubes for reagent dilutions, calibrated pipets, pipet tips, sterile
distilled water, aluminum foil, absorbent paper towels, 1.5 or 2 ml microcentrifuge tubes, and
standard flat bottom microplate (for calibrating vacuum manifold).

5

Assay Workflow

Prewet wells

(for lter 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
or Luminex xPONENT software (see the Run Assay section).

Assay Procedures

Please pay close attention to vortexing, shaking, and incubation times
and to Bio-Plex
specifically for each assay panel.

®

reader PMT (RP1) setting, as these have been optimized

Assay Quick Guide

Each assay kit comes complete with a printed Bio-Plex Pro™ Assay Quick Guide
(bulletin #10031991), which can be used to prepare and run a full 1 x 96-well
assay plate. Users can also download a copy at www.bio-rad.com/bio-plex.

Bead Regions and Multiplexing Compatibility

n

Bead regions for all analytes are listed in the Read Plate section

n

Do not mix analytes between different Bio-Plex panels or reagent kits.

Resulting standard curves and sample values may be inaccurate

™

software

Detailed Instructions

The following pages provide detailed instructions for each step of the
assay procedure, including preparation, running the assay, and reading the
plate with Bio-Plex Manager

7

™

and Luminex xPONENT software.

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
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. Note that Bio-Plex Manager automatically
subtracts the blank (B) MFI value from all other assay wells.

3. User-specified controls, as well as the quality controls supplied in
premixed kits, are assigned to wells in columns 3 and 4.

4. The remainder of the plate is available for samples.

5. Once the total number of wells is known, you can calculate the
required volumes of beads, detection antibody, and streptavidin-PE.
Use Tables 6–7, 9–10, and 11, respectively, or the Calculation
Worksheet on pages 36–37.

Legend

S Standard

™

. A suggested plate layout is shown in Figure 2,

B Blank

X Samples

C Controls

Fig. 2. Suggested plate layout. For detailed instructions on plate
formatting in Bio-Plex Manager, see the Read Plate section.

8

2. Prepare Instrument

These directions are specific for the Bio-Plex® 100/200 reader. To prepare
either a Bio-Plex 3D or Bio-Plex
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. The calibration kit should be run
daily or before each use of the instrument to standardize the fluorescent
signal. For instructions on using other xMAP system software packages,
contact Bio-Rad Technical Support.

The validation kit should be run monthly to ensure optimal performance of
fluidics and optics systems. Refer to either the software manual or online
Help for directions on how to conduct validation.

®

MAGPIX™ reader, consult their respective

™

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
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up
lasers/optics to reach operational temperature.

and follow the instructions. If the system is idle

and wait for the

Calibrate System

1. Select Calibrate and confirm that the default values for CAL1
and CAL2 are the same as the values printed on the bottle of Bio-Plex
calibration beads. Use the Bio-Plex system low RP1 target value.

9

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. Summary of compatible wash stations and plate types.

Wash Method Wash Station Assay Plate

Magnetic separation Bio-Plex Pro Flat bottom plate
Bio-Plex Pro II (use MAG programs)
Bio-Plex

Vacuum filtration Bio-Plex Pro II (use VAC programs) Filter plate
Vacuum manifold (manual)

Setting up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station

The wash station should be primed before use. For more information, refer
to the Bio-Plex Pro Wash Stations Quick Guide (bulletin #5826).

1. Install the appropriate plate carrier on the wash station.

2. Use the Prime procedure to prime channel 1 with 1x wash buffer.

®

handheld magnetic washer

Setting up the Bio-Plex Handheld Magnetic Washer

Place an empty flat bottom plate on the magnetic washer by sliding
it under the retaining clips. Push the clips inward to secure the plate.
Make sure the plate is held securely. If needed, the clips can be adjusted
for height and tension. For detailed instructions, refer to the user guide
(bulletin #10023087).

10

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.

3. To prepare 1x wash buffer, dilute 1 part of 10x stock solution with
9 parts of deionized water.

5. Prepare Standards and Controls

General Instructions

n

It is essential to prepare standards and quality controls (if included)

exactly as described in this section. Incorrect preparation may lead to
low signal or variable measurements from plate to plate

n

The product data sheet provided with the standards lists the most

concentrated point on the standard curve (S1). Enter this information
into Bio-Plex Manager

™

software as instructed in section 9

Using the Quality Controls (optional)

A single vial of quality controls is provided with the 40-plex fixed panel only.
Their use is intended for monitoring the day-to-day quality of assay results.

11