- Bio-Rad
Bio-Plex Pro
TGF-b 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
Principle2
Kit Contents and Storage
4
Recommended Materials
6
Assay Workflow
7
lmportant Considerations
8
Detailed Instructions
1. Plan Plate Layout
9
2. Prepare Instrument
10
3. Prepare Wash Method
11
4. Prepare Standards
12
5. Prepare Samples
15
6. Prepare Coupled Beads
18
7. Run Assay
20
8. Read Plate
25
Troubleshooting Guide
31
Plate Layout Template
36
Calculation Worksheet
37
Safety Considerations
39
Legal Notices
39
Ordering Information
40
Introduction
Transforming Growth Factor (TGF-b) Assays
The TGF-b family of proteins plays an important role in a wide range of
cellular functions including proliferation, differentiation, migration, and
apoptosis. There are three known isoforms of TGF-b with overlapping
functions in normal physiology and in disease states such as cancer.
TGF-b1 promotes TH17 immune cell development and bone growth
and remodeling. TGF-b2 plays a vital role in embryonic development
and has been shown to suppress IL-2 dependent T-cell tumors. TGF-b3
regulates cell differentiation, adhesion, and extracellular matrix formation in
embryogenesis and wound healing.
Bio-Plex Pro™ TGF-b assays are magnetic bead–based multiplex assays
designed to measure TGF-b1, TGF-b2, and TGF-b3 in human, mouse,
and rat sample matrices such as serum, plasma, urine, tissue culture
supernatant, and milk.
Multiplexing with Bio-Plex Pro Assays
Bio-Plex Pro assays enable researchers to quantify multiple protein
biomarkers in a single well of a 96-well plate in just 3 to 4 hours.
These robust immunoassays require as little as 12.5 µl serum or plasma,
or 50 µl cell culture supernatant or 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, productivity, 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:
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Fluorescently dyed 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 types of molecules in a single well of the 96-well
microplate on the Bio-Plex® 3D system, up to 100 different types of
molecules on the Bio-Plex® 200 system, and up to 50 different types
of molecules on the Bio-Plex® MAGPIX™ system
On the Bio-Plex 200 and Bio-Plex 3D systems, a dedicated flow
cytometer with two lasers and associated optics to measure the
different molecules bound to the surface of the beads. In the
Bio-Plex MAGPIX, the entire sample load volume is injected into a
chamber where the beads are imaged using LED and CCD technology
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
Biotinylated
Detection
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. 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
TGF-b1, TGF-b2, and TGF-b3 assays are available in a convenient kit format
that includes assay, reagent, and diluent components in a single box (Table 1).
Table 1. Contents of Bio-Plex Pro™ TGF-b assays.*
1 x 96-Well
Format
Component
Standard diluent
1 bottle, 10 ml
Sample diluent
1 bottle, 40 ml
Assay buffer
1 bottle, 50 ml
Wash buffer
1 bottle, 200 ml
Detection antibody diluent
1 bottle, 5 ml
Streptavidin-PE (100x)
1 tube
Filter and/or flat bottom plate (96-well)
1 plate
Sealing tape
1 pack of 4
Assay Quick Guide
1 booklet
Coupled magnetic beads (20x)
1 tube
Detection antibodies (20x)
1 tube
Standard
1 vial
* Volumes shown are approximate.
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 6 months from the date of purchase
when stored as specified.
4
Reagents Required but Not Supplied
TGF-b is secreted as part of a complex that causes it to be inactive.
It is necessary to expose samples to acidic conditions in order to activate
TGF-b. The following reagents are required:
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1 N hydrochloric acid: To make 100 ml of 1 N HCl, add 8.33 ml of
12 N HCl slowly to 91.67 ml of deionized water, and mix well
1.2 N sodium hydroxide/0.5 M HEPES: To make 100 ml of 1.2
NaOH/0.5 M HEPES, add 12 ml of 10 N NaOH to 75 ml of deionized
water, and mix well. Add 11.9 g of HEPES (free acid, MW 238.3), mix
well, and bring the final volume to 100 ml with deionized water
5
Table 2. Recommended materials.
Item
Ordering Information
Bio-Plex Pro™ Assays Quick Guide 5
Bulletin #10024986 (download
at www.bio-rad.com/bio-plex)
Bio-Plex® 200 system or Luminex system with HTF
Bio-Rad catalog #171-000205
Bio-Plex validation kit
Run the validation kit monthly to ensure optimal performance
of fluidics and optics systems
Bio-Rad catalog #171-203001
Bio-Plex calibration kit
Run the calibration kit daily to standardize fluorescence signal
Bio-Rad catalog #171-203060
Bio-Plex Pro wash station
For use with magnetic bead–based assays only
Bio-Rad catalog #300-34376
Bio-Plex Pro II wash station
For use with both polystyrene (nonmagnetic) and magnetic
bead–based assays
Bio-Rad catalog #300-34377
Bio-Plex handheld magnetic washer
For use with magnetic bead–based assays only
Bio-Rad catalog #170-020100
Bio-Plex Pro flat bottom plates, 40 x 96-well
For magnetic separation on the Bio-Plex Pro wash station
Bio-Rad catalog #171-025001
Microtiter plate shaker
IKA MTS 2/4 shaker for 2 or 4 microplates
or
Barnstead/Lab-Line Model 4625 plate
shaker (or equivalent capable of 300–1,100 rpm)
IKA catalog #320-8000
VWR catalog #57019-600
Bio-Rad® Aurum™ vacuum manifold
For vacuum filtration
Bio-Rad catalog #732-6470
BR-2000 vortexer
Bio-Rad catalog #166-0610
Reagent reservoirs, 25 ml
For capture beads and detection antibodies
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
Reagent reservoir, 50 ml (for reagents and buffers)
VistaLab catalog #3054-1006
Pall Life Science Acrodisc, 25 mm PF syringe filter
(0.8/0.2 µm Supor membrane)
Pall Life Sciences
catalog #4187
Filter plate, 1 x 96-well, clear plastic lid and tray
Bio-Rad catalog #171-304502
Titertube micro test tubes
For preparing replicate standards, samples, and controls
prior to loading the plate
Bio-Rad catalog #223-9390
®
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).
6
Assay Workflow
Prewet wells
(for filter plate only)
Add 50 μl 1x beads to wells
Wash 2 x 100 μl
Add 50 μl standards, blank, activated samples;
incubate 2 hr at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 25 μl 1x detection antibody, incubate
1 hr at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 50 μl 1x streptavidin-PE, incubate
30 min at RT with shaking at 850 rpm
Wash 3 x 100 μl
Resuspend in 125 μl assay buffer,
shake at 850 rpm for 30 sec
Read plate on Bio-Plex system
7
lmportant Considerations
Instruments and Software
The 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.
Assay Procedures
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 includes a printed Bio-Plex Pro™ Assay Quick Guide (bulletin
#10024986), 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
Bead regions for all analytes are listed in the Read Plate section.
Multiplexing Compatibility
Do not mix TGF-b assays with other Bio-Plex assay panels or reagent
kits. Protocols, reagents, and sample treatment conditions are not
compatible.
8
1. Plan Plate Layout
Prior to running the assay, determine the total number of wells in the
experiment using the Plate Layout Template on page 36 or the Plate
Formatting tab in Bio-Plex Manager™ software. 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 and be processed in the same manner as
sample and standard wells. Note that Bio-Plex Manager automatically
subtracts the blank (B) MFI value from all other assay wells.
3.User-defined controls 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, calculate the required
volumes of beads, detection antibody, and streptavidin-PE needed.
Use Tables 7–8, 10–11, and 12, respectively, or the Calculation
Worksheet on pages 37–38.
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 section 8, Read Plate.
9
2. Prepare Instrument
Start up and calibrate the Bio-Plex® 100/200 or similar 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. To prepare either a Bio-Plex® 3D or Bio-Plex®
MAGPIX™ reader, consult its respective user manual.
The validation kit should be run monthly to ensure 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
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
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.
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 even if assays will be run at high RP1.
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.
10
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 Bio-Plex Pro II (use MAG programs)
Bio-Plex ® handheld magnetic washer
Flat bottom plate
Vacuum filtration
Bio-Plex Pro II (use VAC programs)
Vacuum manifold (manual)
Filter plate
Setting up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station
The wash station does not require calibration; however, it should be primed
before use. For more information, refer to the Bio-Plex Pro and Pro II wash
station quick guide (bulletin #5826).
1.Install the appropriate plate carrier on the wash station.
2. Use the prime procedure to prime channel 1 with 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).
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.
11
4. Prepare Standards
General Instructions
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It is essential to prepare standards exactly as described in this section.
Incorrect preparation may lead to low signal or variable measurements
from plate to plate
The peel-off sticker 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 8
Prepare a Diluent for Standards
1. Refer to Table 4 for recommended diluents based on different sample
types. As a general rule, reconstitute and dilute standards in a diluent
similar to the final sample type or sample matrix.
2. If samples are serum or plasma, mix 1 volume of Bio-Plex®
standard diluent with 3 volumes of Bio-Plex sample diluent (each
supplied in the kit). The resulting solution is used for reconstitution
and subsequent dilution of standards. This results in a serum-matrix
based diluent that mimics the matrix in 1:16 diluted serum and
plasma samples.
Table 4. Summary of recommended diluents for standards.
Sample Type
Diluent for Standard Add BSA
Serum and plasma
Culture media, with serum
Culture media, serum-free
Lavage, lysate, other fluids
Standard/sample diluent mix (1:3)
Culture media
Culture media
Sample diluent
None
None
To 0.5% final* (w/v)
To 0.5% final* (w/v)
* At least 0.5% final w/v BSA is recommended to stabilize analytes and reduce adsorption
to labware.
12
Reconstitute a Single Vial of Standards
This procedure prepares enough material to run each dilution in duplicate.
1. Gently tap the vial containing the lyophilized standard.
2. Add 500 μl of the appropriate standard diluent (see Table 4). Do not
use assay buffer to reconstitute the standards.
3. Gently vortex the reconstituted standard for 5 sec, then incubate on
ice for 30 min. Be consistent with the incubation time in every assay
to ensure best results.
4. During the incubation period, prepare the samples as instructed in
section 5, Prepare Samples.
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 new pipet tips for every volume transfer.
1. Label nine 1.5 ml polypropylene tubes S1 through S8 and Blank.
2. Add the specified volume of standard diluent to each tube (Figure 3).
3. V
ortex the reconstituted standards gently for 5 sec before removing
any volume. Add 128 µl to the S1 tube containing 72 µl of standard
diluent. Vortex at medium speed for 5 sec, then use a new pipet tip
to transfer 50 µl from S1 tube to S2 tube. Vortex.
4. Continue with 1:4 (fourfold) serial dilutions from tube S2 to S8 as
shown in Figure 3. Use reconstituted and diluted standards
immediately. Do not freeze for future use.
5. Continue with 1:4 serial dilutions as shown in Figure 3. Use
reconstituted and diluted standards immediately. Do not freeze for
future use.
13
12850505050505050
One
Reconstituted
Standard
72 150150150150150150150150
Transfer Volume, µl
Diluent, µl
S1S2S3S4S5S6S7S8
Blank
Fig. 3. Preparing a fourfold dilution series with a single reconstituted standard.
RP1 (PMT) Setting for Standard Curves
The Bio-Plex® 200 and 3D systems have two RP1 (PMT or photomultiplier
tube) setting options, while the Bio-Plex® MAGPIX™ has no PMT
and therefore no PMT setting options. Instead, MAGPIX uses default
instrument settings similar to low PMT on the Bio-Plex 200 (Table 5).
Table 5. Overview of PMT setting options on Bio-Plex systems.
Instrument
RP1 (PMT)
Bio-Plex 100, 200*
Bio-Plex 3D*
Bio-Plex MAGPIX*
Low, high
Standard, enhanced
N/A, use default instrument settings
* Or similar Luminex-based system.
The Bio-Plex Pro TGF-b assays were developed on the low PMT setting
using the Bio-Plex 200 system. Protocols using alternative standard
dilution series or PMT settings should be validated by the end user.
14
5. Prepare Samples
General guidelines for preparing different sample types are provided here.
For more information, contact Bio-Rad Technical Support.
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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 1.5 or 2 ml polypropylene microcentrifuge
tubes. If a multichannel pipet will be used to load the plate, then aliquot
the required volumes into Titertube® micro test tubes
Do not freeze diluted samples
Important! before running the assay, samples must be activated and
diluted as described on pages 17–18
Serum and Plasma
EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma,
while compatible with Bio-Plex Pro™ assays, may absorb certain soluble
proteins of interest. Avoid using hemolyzed samples as this may lead to
false positive results.
1.Draw whole blood into collection tubes containing anticoagulant.
Invert tubes several times to mix.
2.For serum, allow blood to clot at room temperature for 30–45 min.
For plasma, proceed directly to the centrifugation steps.
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.
5.Activate samples as described below and assay immediately or store
untreated/nonactivated samples in single-use aliquots at –70°C.
Avoid repeated freeze/thaw cycles.
15
Cell Culture Supernatant
1. Collect supernatants and perform centrifugation at 1,000 x g for 15 min
at 4°C. For cell lines cultured in serum-free culture media, collect samples
and add BSA as a carrier protein to a final concentration of at least
0.5% to stabilize protein analytes and to prevent adsorption to labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates are present, centrifuge again at 10,000 x g for 10 min at 4°C.
3. Activate samples as described below and assay immediately or store
untreated/nonactivated samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
Lavage, Sputum, and Other Biological Fluid Samples
Keep all samples on ice until ready for use. The appropriate sample
dilution factor should be determined by the user.
1.Centrifuge at 10,000 x g for 10 min at 4°C to clarify the sample.
2.Activate a portion of the sample as described below and, if needed,
dilute in Bio-Plex® sample diluent with BSA added to a final
concentration of at least 0.5%.
Lysates
Users will need to optimize the lysis, sample dilution, and sample activation
methods to ensure that assay performance is fit for purpose.
1.Prepare the cell or tissue lysates according to the instructions provided
with the Bio-Plex cell lysis kit (catalog #171-304011 or 171-304012).
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. The
lysates should be free of particulate matter.
2.Determine the protein concentration of the lysate. It may be
necessary to test lyse your sample with different volumes of lysing
solution to obtain the specified protein concentration range.
3.Activate lysates as described under Sample Activation and Dilution.
Dilute the activated sample in sample diluent + 0.5% BSA. The
appropriate final lysate protein concentration should be determined
by the user. A starting range to try is 50 to 900 ug/ml.
16
Note: For optimal assay performance, it is important to dilute lysates
as much as possible to reduce the detergent concentration.
4.Store untreated/nonactivated lysates at –20°C to –70°C. Avoid
multiple freeze-thaw cycles
Sample Activation and Dilution
First, prepare samples as described above. To measure immunoreactive
TGF-b, it is necessary to treat all sample types with the following activation
procedure. Samples should be assayed immediately after the neutralization
step. Do not activate the TGF-b standards.
Table 6. Summary of recommended sample diluents and dilution factors.
Sample Type
Diluent
Add BSA
Sample Dilution
Serum and plasma
Culture media, with serum
Culture media, serum-free
Lavage, lysate, other fluids
Sample diluent
Culture media
Culture media
Sample diluent
None
None
To 0.5% final**
To 0.5% final**
1:16 final*
User optimized
User optimized
User optimized
* For example, activate 25 μl sample, neutralize, and bring to a final volume of 400 μl.
**At least 0.5% final w/v BSA is recommended to stabilize analytes and reduce adsorption to
labware.
Activation of Serum and Plasma
1.To activate the sample, add 1 volume of acid (1 N HCl) to 5 volumes
of sample. For example, add 5 μl acid to 25 μl of sample. Mix
thoroughly and incubate for 10 min at room temperature.
2.To neutralize the sample, add a volume of base (1.2 N NaOH/0.5 M
HEPES) equal to the volume of 1 N HCl used. In this example, add
5 μl base and mix thoroughly. Treated sample volume is now 35 μl.
3.The recommended dilution is 1:16 of the starting (untreated) sample
volume. In this example, starting sample volume was 25 μl and a 1:16
dilution gives 400 μl. To reach a final volume of 400 µl, add 365 μl
Bio-Plex sample diluent to 35 μl treated sample.
Note: To achieve neutral pH of a sample (pH 7.2 to 7.6), the actual volume
of base required may vary depending on initial sample pH and the buffering
capacity of the sample. Verify pH using pH paper before running the assay.
17
Activation of Cell Culture Supernatant and Other
Biological Fluids
Samples may be run “neat” after activation/neutralization or be diluted as
required. The appropriate dilution factor should be optimized by the user.
Ensure a final sample volume after treatment and dilution of at least 125 µl
to allow for duplicate wells on the assay plate.
1.For example, if a 1:4 dilution is desired, activate the sample by
adding 10 µl acid to 50 µl sample. Mix thoroughly and incubate for
10 min at room temperature.
2.To neutralize sample, add 10 µl base. Mix thoroughly. Treated sample
volume is now 70 μl.
3. Dilute to 1:4 final in the same diluent used to prepare the standards.
In this example, starting sample volume was 50 μl and a 1:4 dilution
gives 200 μl. To reach a final volume of 200 μl, add 130 μl diluent to
70 μl treated sample
Note: Serum-containing culture medium may contain high concentrations
of TGF-b. A preliminary measurement of medium alone is recommended
to determine baseline levels.
6. Prepare Coupled Beads
Instructions are provided for diluting the coupled beads to a 1x concentration.
1.Use Tables 7–8 or the Calculation Worksheet on pages 37–38 to
calculate the volume of coupled beads and assay buffer needed.
2.Add the required volume of Bio-Plex assay buffer to a 15 ml
polypropylene tube.
3. Vortex the stock 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.
18
4.Dilute coupled beads to 1x by pipetting the required volume into the 15 ml
tube. Vortex.
Each well of the assay plate requires 2.5 μl (20x stock) adjusted to a
final volume of 50 μl in assay buffer.
5. Protect the beads from light with aluminum foil. Equilibrate to room
temperature prior to use.
Note: To minimize volume loss, use a 200–300 μl capacity pipet
to remove beads from the stock tube. If necessary, perform the
volume transfer in two steps. Do not use a 1,000 μl capacity pipet
and/or a wide bore pipet tip.
Preparing 1x coupled beads from 20x stock (includes 20% excess volume)
Table 7. Premixed panel or one singleplex assay.
# of Wells
20x Beads, µl Assay Buffer, µl Total Volume, µl
96
288
5,472
5,760
48
144
2,736
2,880
Table 8. Mixing singleplex assays.
# of Wells
20x Beads, µl Singleplex #1
20x Beads, µl Singleplex #2
Assay Buffer, µl Total Volume, µl
96
288
288
5,184
5,760
48
144
144
2,592
2,880
19
7. Run Assay
Considerations
n
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Bring all assay components and samples to room temperature before use
Use calibrated pipets and pipet carefully, avoiding bubbles. Use new
pipet tips for every volume transfer
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 in the dark on a shaker at
850 ± 50 rpm. Cover the plate with sealing tape and protect from
light with aluminum foil
Table 9. Summary of wash options and protocols. After each assay step, select the
appropriate Bio-Plex Pro™ wash station program or perform the appropriate manual wash step
as summarized below.
Bio-Plex Pro or Pro II Wash Station
Bio-Plex Pro II
Wash Station Handheld Magnet
or Vacuum Manifold
Assay Step
Manual Wash Steps
Magnetic Program
Vacuum Program
Add beads to plate
MAG x2
VAC x2
2 x 100 μl
Sample incubation
Detection Ab incubation
SA-PE incubation
MAG x3
MAG x3
MAG x3
VAC x3
VAC x3
VAC x3
3 x 100 μl
3 x 100 μl
3 x 100 μl
Considerations when Using a Vacuum Manifold
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After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
To wash, add 100 μl wash buffer to each well and remove the liquid as
before. Ensure that all wells are exposed to the vacuum
Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
Place the assay plate on the plastic plate holder/tray as needed
Before each incubation, gently cover the plate with a new sheet of
sealing tape. Avoid pressing down over the wells to prevent leaking
from the bottom
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20
Add Coupled Beads, Standards, and Samples
1. Cover unused wells with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat bottom plate.
Prewet the wells using 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) coupled beads for 30 sec at medium speed.
Pour the diluted coupled beads into a reagent reservoir and transfer
50 µl to each well of the assay plate.
Tip: A multichannel pipet is highly recommended for ease of use
and efficiency.
4.Wash the plate two times with 100 µl Bio-Plex® wash buffer using
the wash method of choice.
5.Gently vortex the diluted standards, blanks, samples, and controls (if applicable) for 5 sec. Transfer 50 µl to each well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover plate with a new sheet of sealing tape and protect from light
with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 2 hr at
room temperature (RT).
Note: 850 rpm provides equivalent performance to shaker settings
recommended in previous manuals (1,100 rpm for 30 sec, 300 rpm for
incubation).
Prepare and Add Detection Antibodies
Instructions are provided for diluting the detection antibodies to a
1x concentration.
1. While the samples are incubating, use Tables 10–11 or the
Calculation Worksheet on pages 37–38 to calculate the volume
of detection antibodies and detection antibody diluent needed.
Detection antibodies should be prepared 10 min before use.
21
2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the stock detection antibodies for 15–20 sec at medium
speed, then perform a 30 sec spin to collect the entire volume at the
bottom of the tube.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube.
Each well of the assay requires 1.25 μl (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 10. Premixed panel or one singleplex assay.
# of Wells
20x Detection Antibodies, µl
Detection Antibody
Diluent, µl
Total Volume, µl
96
150
2,850
3,000
48
75
1,425
1,500
Table 11. Mixing singleplex assays.
# of Wells
96
48
20x Detection Antibodies, µl
Singleplex #1
20x Detection Antibodies, µl
Singleplex #2
150
75
Detection
Antibody
Diluent, µl Total Volume, µl
150 2,7003,000
75
1,350
1,500
5. After incubating the beads, samples, standards, and blank, slowly
remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer.
7. Vortex the diluted (1x) detection antibodies gently for 5 sec. Pour
into a reagent reservoir and transfer 25 μl to each well using a
multichannel pipet.
8. C
over plate with a new sheet of sealing tape and protect from light with
aluminum foil. Incubate on shaker at 850 ± 50 rpm for 1 hr at room
temperature.
22
Prepare and Add Streptavidin-PE (SA-PE)
1.While the detection antibodies are incubating, use Table 12 or the
Calculation Worksheet on pages 37–38 to calculate the volume of
SA-PE (100x) and assay buffer needed. Streptavidin-PE should be
prepared 10 min before use.
2.Add the required volume of assay buffer to a 15 ml polypropylene
tube.
3. Vortex the 100x 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 (100x stock) adjusted to a final
volume of 50 µl in assay buffer.
Table 12. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
# of Wells
100x SA-PE, µl
Assay Buffer, µl
Total Volume, µl
96
60
5,940
6,000
48
30
2,970
3,000
5. After the detection antibody incubation, slowly remove and discard
the sealing tape.
6. Wash the plate three times with 100 µl wash buffer.
7. V
ortex the diluted (1x) SA-PE at medium speed for 5 sec. Pour
into a reagent reservoir and transfer 50 µl to each well using a
multichannel pipet.
8. Cover plate with a new sheet of sealing tape and protect from light
with aluminum foil. Incubate on shaker at 850 ± 50 rpm for 30 min
at room temperature.
23
9. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
10. Wash the plate three times with 100 µl wash buffer.
11.To resuspend beads for plate reading, add 125 µl of assay buffer to
each well. Cover the plate with a new sheet of sealing tape. Shake at
room temperature at 850 ± 50 rpm for 30 sec, and slowly remove the
sealing tape. Ensure that the plate cover has been removed before
placing the plate on the reader.
12.Remove the sealing tape and read the plate using the settings
below. Refer to section 8, Read Plate, for details.
Note: Reading at alternative PMT settings on the Bio-Plex 100,
Bio-Plex 200, or Bio-Plex 3D requires validation by the end user to
ensure that results meet the user’s acceptance criteria.
Table 13. Read 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 Bio-Plex® MAGPIX™* N/A, use default instrument settings * Or similar Luminex-based system.
24
50
Default
8. 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. For instructions using other xMAP system software
packages, contact Bio-Rad Technical Support or your regional Bio-Rad
field applications specialist.
Prepare Protocol in Bio-Plex Manager
Software v 6.0 and Higher
The protocol should be prepared in advance so that the plate is read as
soon as the experiment is complete.
A protocol file specifies the analytes used in the reading, the plate wells
to be read, sample information, the values of standards and controls, and
instrument settings.
Bio-Plex Manager software contains protocols for most Bio-Plex® assays.
Choose from available protocols or create a new protocol. To create a new
protocol, select File, then New from the main menu. Locate and follow the
steps under Protocol Settings.
1.Click Describe Protocol and enter information about the
assay (optional).
2.Click Select Analytes and create a new panel. Visually confirm the
selected analytes and proceed to step 3.
a.
Click the Add Panel button
in the Select Analytes toolbar.
Enter a new panel name. Select Bio-Plex Pro Assay Magnetic
from the assay pull-down menu. If using Bio-Plex Manager version
5.0 or lower, select MagPlex from the assay pull-down menu.
b.
Click the Add button. Enter the bead region number and name
for the first analyte. Click Add Continue to repeat for each
analyte in the assay.
25
For reference, bead regions are shown in Table 14.
c. Click the Add button when the last analyte has been added and
click OK to save the new panel.
d. Highlight analytes from the Available list (left) and move to the
Selected list (right) using the Add button. To move all analytes at
once, simply click the Add All button.
e.
If some of the analytes need to be removed from the Selected
list, highlight them and select Remove. If desired, it is possible to
rename the panel by clicking on Rename Panel and entering a
new panel name.
Table 14. TGF-b assay bead regions.
Analyte
Bead Region
TGF-b113
TGF-b272
TGF-b366
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. Repeat this process for
blanks B , controls C , and samples X .
4.Click Enter Standards Info in the Protocol Settings bar.
a. Enter the highest concentration of each analyte in the top row
(labeled S1) of the table. S1 concentration information is included
on the peel-off sticker provided with each vial of standards.
26
Fig. 4. Plate formatting.
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, and for user-defined controls, select
an analyte from the pull-down menu, then enter a description and
concentration. Repeat for each additional analyte in the assay.
6. Click Enter Sample Info and enter sample information and the
appropriate dilution factor.
27
7. Click Run Protocol and confirm that the assay settings are correct.
a. Refer to Table 13 for the recommended RP1 (PMT) setting.
Protocols using alternative PMT settings should be validated by
the end user.
b. Confirm data acquisition is set to 50 beads per region. In
Advanced Settings, confirm that the bead map is set to 100
region, the sample size is set to 50 μl, and the DD gates are set to 5,000 (Low) and 25,000 (High). In Bio-Plex Manager software versions 4.0, 4.1, and 4.1.1, check Override Gates
and set the DD gate values as indicated.
c. Select Start, name and save the .rbx file, and begin data
acquisition. The Run Protocol pop-up screen will appear. Click
Eject/Retract to eject the plate carrier.
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 — 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.If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate (if HTF are not present).
Select Wash Between Plates and follow the instructions. Then
repeat the Prepare Protocol and Acquire Data instructions.
5. When data acquisition is complete, select Shut Down
follow the instructions.
28
and
Reacquire Data
It is possible to acquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in the Run Protocol step.
Any previous data will be overwritten.
1. Check the wells from which data will be reacquired.
2. Remove the buffer with the wash method of choice.
3.Add 100 µl assay buffer to each well. Cover the filter plate with a
new sheet of sealing tape. Shake the plate at 850 ± 50 rpm for
30 sec. Slowly remove the sealing tape before placing the plate on
the plate reader.
4. Repeat the Acquire Data steps to reacquire data. The data acquired
should be similar to those acquired initially; however, the acquisition
time will be extended because the wells have fewer beads.
Data Analysis: Removing Outliers
Outliers are identified as standard data points that do not meet accuracy
or precision requirements and should be considered invalid when
performing curve fitting. As such, they should be removed to generate a
more realistic and accurate standard curve. This may result in an extended
assay working range and allow quantitation of samples that might
otherwise be considered out of range (OOR).
In Bio-Plex Manager software version 6.0 and higher, outliers can be
automatically removed by selecting the Optimize button in the Standard
Curve window. In Bio-Plex Manager software 6.0 and earlier versions,
outliers can be manually selected in the Report Table. Visit online Help to
learn more about the standard curve optimizer feature and how outliers
are determined.
Previous Versions of Bio-Plex Manager Software
For instructions on using previous versions of Bio-Plex Manager software,
please contact Bio-Rad Technical Support.
29
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. Select MagPlex as the bead type for magnetic beads, which
automatically sets the DD gates.
2. Volume = 50 µl.
3.Refer to Table 13 to select the appropriate PMT setting for
your instrument.
4. Plate name: 96-well plate.
5. Analysis type: Quantitative; 5PL Curve Fit.
6. Number of standards: 8.
Select Analytes to set up the panel.
1. Enter pg/ml in the Units field.
2. Enter 50 in the Count field.
3. Select the bead region and enter the analyte name.
4. Click Apply all for Units and Count.
Select Stds and Ctrls.
1. Enter standard concentrations, lot number, dilution factor, and other
information as applicable.
After the assay is complete, select Results, then select Saved Batches.
30
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 determine if the array
reader is functioning properly.
Possible Causes
Standards were not reconstituted
consistently between assays
Possible Solutions
Reconstituted standards and
diluted samples were not stored
properly
Reconstituted standards and diluted
samples should be prepared on ice
as instructed. Prior to plating, the
reconstituted standards and diluted
samples should be equilibrated to
room temperature.
Bottom of filter plate not dry
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent cross-well
contamination.
High Inter-Assay CV
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
31
Possible Causes
Possible Solutions
High Intra-Assay CV
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.
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.
Contamination with wash buffer
during wash steps
During the wash steps, be careful
not to splash wash buffer from one
well to another. Be sure that the
wells are filtered completely and that
no residual volume remains. Ensure
that the microplate shaker setting is
not too high. Reduce the microplate
shaker speed to minimize splashing.
Slow pipetting of samples and
reagents across the plate
Sample pipetting across the entire
plate should take less than 4 min.
Reagent pipetting across the entire
plate should take less than 1 min.
Bio-Plex wash station:
insufficient washing due to
clogged pins
Clean dispensing pins with the thicker
of the 2 cleaning needles provided
with washer. Perform regular rinses to
minimize salt buildup.
32
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 30 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.
Filter plate not shaken enough
before incubation steps and prior
to reading
Shake the filter plate at
850 ± 50 rpm for 30 sec before
incubation steps and immediately
before reading the plate.
Reader is clogged
Refer to the troubleshooting guide
in the Bio-Plex system hardware
instruction manual (bulletin
#10005042).
Low Signal or Poor Sensitivity
Standards reconstituted
incorrectly
Follow the standard preparation
instructions carefully.
Detection antibody or streptavidin-PE diluted incorrectly
Check your calculations and be
careful to add the correct volumes.
33
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.
High Background Signal
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.
Poor Recovery
Expired Bio-Plex reagents
were used
Check that reagents have not
expired. Use new or nonexpired
components.
Incorrect amounts of components
were added
Check your calculations and be
careful to add the correct volumes.
Microplate shaker set to an
incorrect speed
Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may
cause splashing and contamination.
Use the recommended plate shaker.
34
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. If assay drift is suspected, retest
the samples by positioning them next to the
standards. If contamination of standards
is suspected, check the standard replicate
value and be careful when adding samples to
the wells. Matrix effects could also produce
negative sample values.
Bio-Plex Manager™ software automatically
subtracts the blank (B) FI value from all other
assay wells. While this has no impact on
observed concentrations of samples within the
assay working range, it may result in a negative
FI value if the blank’s FI value is greater than
either the standard or sample value. If this is
undesirable, then assign wells as a sample (X) or
control (C) in the protocol or results file.
Poor precision in serum
and plasma sample
measurements
Check if any interfering components such as
heparin-based anticoagulant, additives, or
gel from separators were introduced into the
samples. Avoid using hemolyzed and heavily
lipemic samples. Remove visible particulate
in samples by centrifugation. Avoid multiple
freeze/thaw cycles of samples.
35
Plate Layout Template
36
Calculation Worksheet
If using either a premixed panel or one singleplex assay with 20x stocks of beads
and detection antibodies, 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
12
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
23
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
234
d. Volume of 20x coupled beads required: _______ µl /20 = _______ µl
45
e. Volume of assay buffer required: _______ µl – _______ µl = _______
456
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): _______ x 25 µl = _______ µl
17
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
78
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
78 9
d. Volume of 20x detection antibodies required: _______ µl /20 = _______ µl
910
e. Volume of detection antibody diluent required: _____ µl – _____ µl = _____ µl
91011
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
112
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
1213
c. Total volume of 1x streptavidin-PE: ______ µl + ______ µl = ______ µl
121314
d. Volume of 100x streptavidin-PE required: _______ µl / 100 = _______ µl
1415
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
14 1516
37
If mixing singleplex assays with 20x stocks of beads and detection antibodies, 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
12
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
23
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
234
d. Enter the number of singleplex sets (or analytes) that will be multiplexed = _______
5
e. Volume of 20x coupled beads required from each stock tube:
_______ µl / 20 = _______ µl
46
f. Total volume of combined bead stocks: _______ x _______ µl = _______ µl
56
7
g. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
478
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): _______ x 25 µl = _______ µl
19
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
910
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
9
1011
d. Enter the number of singleplex sets (or analytes) that will be multiplexed = _______
5
e. Volume of 20x detection antibodies required from each stock tube:
_______ µl / 20 = _______ µl
1112
f. Total volume of combined detection antibody stock: _____ µl x _____ = _____ µl
12 513
g. Volume of detection antibody diluent required: ____ µl – ____ µl = ____µl
111314
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
115
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
1516
c. Total volume of 1x streptavidin-PE: ______ µl + ______ µl = _______ µl
1516 17
d. Volume of 100x streptavidin-PE required: _______ µl / 100 = _______ µl
1718
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
1718 19
38
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
(U.S. government publication: Biosafety in Microbiological and Biomedical
Laboratories (CDC, 1999)).
Legal Notices
Acrodisc, Acroprep, and Supor are trademarks of Pall Corporation.
MagPlex, xMAP, xPONENT, FLEXMAP 3D, and Luminex are trademarks of
Luminex Corporation.
The Bio-Plex suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.
39
Ordering Information
TGF-b Premixed Multiplex Panel Bio-Plex Pro™ TGF-b 3-plex Panel, 1 x 96
Catalog #
171-W4001M
TGF-b Singleplex Sets* Bio-Plex Pro TGF-b1 Set, 1 x 96 Bio-Plex Pro TGF-b2 Set, 1 x 96
Bio-Plex Pro TGF-b3 Set, 1 x 96 Catalog #
171-V4001M
171-V4002M
171-V4003M
Premixed Cytokine Panels
Catalog #
Bio-Plex Pro Human Cytokine 8-Plex Panel, 1 x 96
M50-000007A
Bio-Plex Pro Human Cytokine 17-Plex Panel, 1 x 96
M50-00031YV
Bio-Plex Pro Human Cytokine 21-Plex Panel, 1 x 96
MF0-005KMII
Bio-Plex Pro Human Cytokine 27-Plex Panel, 1 x 96
M50-0KCAF0Y
Bio-Plex Pro Human Cytokine Th1/Th2 Panel, 1 x 96
M50-00005L3
Bio-Plex Pro Mouse Cytokine 8-Plex Panel, 1 x 96
M60-000007A
Bio-Plex Pro Mouse Cytokine 9-Plex Panel, 1 x 96
MD0-00000EL
Bio-Plex Pro Mouse Cytokine 23-Plex Panel, 1 x 96
M60-009RDPD
Bio-Plex Pro Mouse Cytokine Th1/Th2 Panel, 1 x 96
M60-00003J7
Bio-Plex Pro Mouse Th17 Cytokine Panel A 6-Plex, 1 x 96
M60-00007NY
Bio-Plex Pro Mouse Th17 Cytokine Panel B 8-Plex, 1 x 96
171-FA001M
Bio-Plex Pro Mouse Cytokine Th1 Panel, 1 x 96 L60-00004C6
Bio-Plex Pro Mouse Cytokine Th2 Panel, 1 x 96 L60-000UKVT
Bio-Plex Pro Rat Th1/Th2 Panel, 1 x 96
171-K1002M
Bio-Plex Pro Rat Cytokine 24-Plex Panel, 1 x 96
171-K1001M
* Require reagent kit (#171-304070 for vacuum separation or #171-304070M for magnetic
separation) and a vial of standards (#171-X40001).
Bio-Plex® x-Plex™ Assays (We Mix)
Premium custom assay service using the Bio-Plex Assay Builder,
www.bio-rad.com/bio-plex/assaybuilder, to select analytes and plate
type of interest. Assays are supplied as premixed coupled beads and
detection antibodies in the all-in-one kit format.
Bio-Plex Express Assays (You Mix)
Fast and economical custom assay service using the Bio-Plex Assay
Builder, www.bio-rad.com/bio-plex/assaybuilder, to select analytes and
plate type of interest. Assays are supplied as individual sets of coupled
beads and detection antibodies in the all-in-one kit format.
Singleplex Sets and Individual Components
A host of singleplex sets and individual assay components are available. For
more information, refer to bulletin 5507 or go to www.bio-rad.com/bio-plex.
40
Bio-Rad
Laboratories, Inc.
Life Science
Group
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800
Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0
Greece 30 210 9532 220 Hong Kong 852 2789 3300
Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3188
South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 800 88 22 88
United Kingdom 020 8328 2000
10024984 Rev A
Sig 1212
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