Bio-Rad Human Inflammation Assays User Manual

Bio-Plex Pro

™

Human

Inflammation 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

1.1. Plan Plate Layout 8

2.2. Prepare Instrument 9

3.3. Prepare Wash Method 10

4.4. Preparing Buffer and Diluent 11
5 5. Prepare Standards and Controls 11

6. Prepare Samples 13

7.7. Prepare Coupled Beads 17
8 8. Run Assay 18
9 9. Read Plate 22

Troubleshooting Guide 29

Plate Layout Template 34

Calculation Worksheet 35

Safety Considerations 37

Legal Notices 37

Ordering Information 38

Introduction

Cytokines are low molecular weight proteins that are generated mostly
by immune cells and in turn play crucial roles in a cascade of vascular
changes, including the differentiation, maturation, and activation of various
immune cells. These cytokines may exert either pro- or anti-inflammatory
effects, depending on the nature of the activating or inhibitory signal and
timing. Inflammatory processes resulting from the secretion of these
cytokines lead to various manifestations in skin, joints, and other tissues,
as well as altered cytokine homeostasis. These processes are required for
a healthy immune response. However, they are also responsible for many
pathological conditions, such as autoimmune, neurological, cardiovascular,
and metabolic disorders, as well as tumor proliferation. Chief among
the immune cells are the regulatory T cells (Tregs), which are known to
modulate the immune system by maintaining tolerance to self-antigens and
limiting autoimmune and chronic inflammatory diseases.

The Bio-Plex Pro™ human inflammation panel I consists of a wide range
of assays to facilitate the study of pro- and anti-inflammatory cytokines
in various pathophysiological conditions. These assays provide a tool to
study the impact of Tregs on clinical diseases, to evaluate the mechanism
of action of investigational agents. The assays can also facilitate the
drug discovery process by studying efficacy and toxicity. The assays
are compatible with a wide variety of sample matrices, including serum,
plasma, and culture supernatant. For researchers working with limited
sample volume, the capacity to multiplex will provide an effective option
over the traditional ELISA method.

Multiplexing with Bio-Plex Pro Human Inflammation
Assays

These 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 μl of serum or plasma or 50 μl
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 with 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® 3D system, up to
100 different molecules on the Bio-Plex® 200 system, and up to 50
different molecules on the Bio-Plex® MAGPIX™ system

n

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

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.

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™ software presents data as median fluorescence
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™ software, data from multiple instrument runs
can be combined into a single project for easy data management, quick
visualization of results, and simple statistical analysis.

3

Kit Contents and Storage

Reagents Supplied

Bio-Plex Pro™ human inflammation 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 (10x)

Detection antibodies (10x)

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 premixed panel only.
** Volumes shown are approximate.

Quantity

1 bottle (30 ml)

1 bottle (3.5 ml)

1 bottle (10 ml)

1 bottle (50 ml)

1 bottle (60 ml)

1 pack of 4

**

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 Inflammation Assays Quick Guide

Bio-Plex® MAGPIX™, Bio-Plex Manager

™

Ordering Information

Bulletin #10044281 (download
at www.bio-rad.com/bio-plex)

Bio-Rad catalog #171-015001

Bio-Plex validation kit

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 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)

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)

Bio-Rad catalog #166-0610

VistaLab catalog #3054-1002

or

VistaLab catalog #3054-1004

VistaLab catalog #3054-1006

Pall Life Sciences

catalog #4187

Other: 5 or 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

Prewet wells

(for lter plate only)

Assay Workflow

Add 50 μl 1x beads to wells

Add 50 μl standards, samples, controls;

incubate on shaker at 850 rpm for 1 hr at RT

Add 25 μl 1x detection antibody; incubate

on shaker at 850 rpm for 30 min at RT

Add 50 μl 1x streptavidin-PE; incubate

on shaker at 850 rpm for 10 min at RT

Resuspend in 125 μl assay buffer;

Wash 2 x 100 μl

Wash 3 x 100 μl

Wash 3 x 100 μl

Wash 3 x 100 μl

shake at 850 rpm for 30 sec

Acquire data on Bio-Plex system

Notes on sample preparation: Once thawed, keep samples on ice.
Prepare dilutions just prior to the start of the assay and equilibrate to room
temperature before use.

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).

Assay Procedures

Please 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.

Assay Quick Guide

Each assay kit comes complete with a printed Bio-Plex Pro™ Assay Quick Guide
(bulletin #10044281), 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. This
kit uses Bio-Plex Pro reagent kit III.

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™ and Luminex xPONENT software.

7

1. Plan Plate Layout

Determine the total number of wells in the experiment using the Plate
Layout Template on page 34 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 HB. 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 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 35–36.

Legend

S Standard

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® 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. 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.

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
fluidics 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 and follow the instructions. If the system is idle
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up and wait for the
lasers/optics to reach operational temperature.

9

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.

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® handheld magnetic washer

Setting Up the Bio-Plex Pro 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.

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

4. Preparing Buffer and Diluent

Prepare Wash Buffer

1. Bring the 10x stock solution to room temperature.

2. If crystals are present, 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 10x stock solution with
9 parts 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 control is provided with the premixed panels only. Their use
is intended for monitoring the day-to-day quality of assay results.

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.

11