Bio-Rad Bio-Plex Pro Magnetic Cell Signaling Assays User Manual
Bio-Plex Pro™
Cell Signaling 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 Components and Storage 4
Recommended Materials 5
Assay Workflow 6
lmportant Considerations 7
Detailed Instructions 8
11. Prepare the Samples 8
22. Plan the Plate Layout 12
33. Prepare the Wash Method 14
44. Run the Assay 16
55. Read the Plate 21
Troubleshooting Guide 28
Plate Layout Template 31
Calculation Worksheet 32
Safety Considerations 34
Legal Notices 34
Ordering Information 35
Introduction
Bio-Plex Pro™ Cell Signaling Assays
Cell signaling is a complex process through which cells receive and
respond to stimuli from the surrounding environment. For example,
circulating cytokines and chemokines elicit a response from lymphocytes
by binding to cell surface receptors and activating intracellular
phosphoprotein signaling cascades. This turns on and off specific
genes in the nucleus — thus regulating protein expression, cell growth,
proliferation, motility, and survival (Chang and Karin 2001). Aberrant
signaling can lead to serious pathologies including cancer, autoimmune
diseases, cardiovascular disease, and neurological disorders.
Understanding which cell types and signaling pathways are involved in a
disease allows researchers to develop more precisely targeted therapies
with better efficacy and safety.
Bio-Plex Pro cell signaling assays are magnetic bead-based
immunoassays for the detection of intracellular phosphoproteins and
total target proteins in cell and tissue lysates. The assays are available as
singleplex sets, which researchers can combine on their own to make a
multiplex assay, or as premixed, all-in-one, multiplex kits. Phosphoprotein
detection and total target detection are carried out in separate wells of a
96-well assay plate, with just 1–10 μg of sample per well.
The assays have been optimized for exceptional sensitivity, high
specificity and improved performance over western blotting. The use
of magnetic (MagPlex) beads allows automation of wash steps on a
Bio-Plex Pro or similar wash station, which greatly simplifies assay
processing and improves assay precision.
For a complete list of all Bio-Plex Pro cell signaling targets, please visit
www.bio-rad.com/bio-plex.
References
Chang L and Karin M (2001). Mammalian MAP kinase signalling
cascades. Nature 410, 37–40.
1
Principle
Technology
The Bio-Plex
®
suspension array system is built upon the three core
elements of xMAP technology:
n
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
Bio-Plex
Bio-Plex
n
A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems are
®
®
®
3D system, up to 100 different types of molecules on the
200 system, and up to 50 different types of molecules on the
MAGPIX™system
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 entire sample load volume 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,
which 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). The relative concentration of analyte bound
to each bead is proportional to the MFI of the 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 Components and Storage
Table 1. Components required for a complete 1 x 96-well cell signaling assay.
Note that custom x-Plex
detection antibodies in an all-in-one kit.
Component
Singleplex* or Multiplex Set
Coupled magnetic beads (20x)
Detection antibodies (20x)
Cell Signaling Reagent Kit (catalog #171-304006M)
Cell wash buffer
Cell lysis buffer
Cell lysis factor QG
Wash buffer
Detection antibody diluent
Resuspension buffer
Streptavidin-PE (100x)
Flat bottom plate
Sealing tape
Assay Quick Guide
Bio-Rad Cell Lysate Controls (optional)**
Positive control (treated or untreated)
Negative control (phosphatase treated)
* Users can mix compatible singleplex sets to create their own multiplex assays.
** Refer to Table 3 to identify the appropriate controls for your phosphoprotein or total target
of interest.
*** Volumes shown are approximate.
™
assays include a premixed (multiplex) set of beads and
Quantity
1 tube
1 tube
1 bottle (50 ml)
1 bottle (50 ml)
1 vial
1 bottle (330 ml)
1 bottle (10 ml)
1 bottle (40 ml)
1 tube
1 plate
1 pack of 4
1 booklet
50 μg per vial
50 μg per vial
***
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
Bio-Plex
Assays Quick Guide 3
®
200 system or Luminex system with HTF
Bio-Plex validation kit
Run monthly
Bio-Plex calibration kit
Run daily to standardize fluorescence signal
Bio-Plex Pro wash station
Ordering Information
Bulletin #10024930 (download
at www.bio-rad.com/bio-plex)
Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001
Bio-Rad catalog #171-203060
Bio-Rad catalog #300-34376
For use with magnetic bead-based assays only
Bio-Plex handheld magnetic washer
Bio-Rad catalog #171-020100
For magnetic bead–based assays only
Bio-Plex Pro flat bottom plates (forty 96-well plates)
Bio-Rad catalog #171-025001
For magnetic separation on the Bio-Plex Pro wash station
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: 32 mm PF syringe filter
(0.45 µm Supor membrane)
™
protein assay kit II
DC
Phenylmethylsulfonyl fluoride (PMSF)
Dimethyl sulfoxide (DMSO)
Kontes tissue grinder
Bio-Rad catalog #166-0610
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
VistaLab catalog #3054-1006
Pall Life Sciences
catalog #4654
Bio-Rad catalog #500-0112
Sigma catalog #P7626
Sigma catalog #D2650
VWR catalog #KT885000-0002
Other: Polypropylene tubes for reagent dilutions, calibrated pipets, pipet tips, sterile distilled
water, aluminum foil, paper towels, 1.5 or 2 ml microcentrifuge tubes, and a 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 2x
Add 50 μl samples, controls, and blanks;
incubate overnight at RT with shaking
Wash 3x
Add 25 μl 1x detection antibody,
incubate 30 min at RT with shaking
Wash 3x
Add 50 μl 1x s treptavidin- PE,
incubate 10 min at RT with shaking
Wash 3x
Add 125 μl of res uspens ion buffer,
shake for 30 sec
Acquire data
6
lmportant Considerations
Assay Procedures
n
Please pay close attention to vortexing, shaking, and incubation
times and to Bio-Plex
optimized specifically for the cell signaling assays
n
For optimal performance, use only reagents specific for Bio-Plex Pro™
cell signaling assays. Reagents in other Bio-Plex assay panels have not
been validated for use in the cell signaling assays
n
Do not reuse diluted (1x) coupled beads, detection antibodies, or
streptavidin-PE
n
Wash as outlined in Table 5. Incomplete washes may cause
assay variation
n
If the data are not acquired immediately, the assay plate may be stored
at 4°C for up to 24 hr protected from light
Assay Quick Guide
Each assay kit comes complete with a printed Bio-Plex Pro Assay Quick Guide
(bulletin 10024930), 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 Table 12
n
Compatible singleplex assays may be mixed to create a multiplex assay
n
Do not mix phosphoprotein assays with corresponding total target
assays (for example, phospho-Akt and total Akt)
®
reader PMT (RP1) setting, as these have been
Instruments and Software
The Bio-Plex Pro cell signaling 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 (Section 5,
Read the Plate, under Detailed Instructions).
7
Detailed Instructions
The following pages provide detailed instructions for each step of the assay
procedure, including sample preparation, running the assay, and reading
the plate with Bio-Plex Manager
™
and Luminex xPONENT software.
1. Prepare the Samples
Considerations
n
The degree of phosphorylation of a given analyte is highly dependent
on the cell type and cell stimulation or treatment conditions
n
Cell lines may vary in their signaling responses to the same stimulation
n
The suggested final protein concentration range in the assay is
3–200 μg/ml (0.15–10 μg per assay well) except for Pl3K p85 (Tyr
which is 31–1,000 μg/ml (1.6–50 µg/well)
n
Optimization of cell lysate concentration may be needed based on
target protein expression levels
n
Cell lysate should be clear of particulate matter before use
Cell Lysates
The Bio-Plex Pro™ cell signaling reagent kit (catalog #171-304006M)
is required for preparing lysates derived from cell culture and tissue
samples. Just before use, prepare an adequate volume of cell lysis buffer
by adding PMSF and cell lysis factor QG.
n
Prepare 500 mM PMSF by dissolving 0.436 g PMSF in 5 ml DMSO.
(Store as aliquots at –20°C). Add PMSF to the cell lysis buffer at a final
concentration of 2 mM
n
Reconstitute cell lysis factor QG to 100x with 250 μl of diH2O and vortex
to mix. Add the reconstituted factor to the cell lysis buffer to a final 1x
working concentration
458
)
8
Adherent Cells
1. Stop the treatment reaction by aspirating the culture medium and
quickly rinsing the cells with ice-cold cell signaling cell wash buffer
(bottle with the blue cap). The volume of buffer required is the same
as the volume of aspirated cell culture medium. Keep the cells on ice
during all steps when possible.
2. Completely remove the buffer before lysing the cells.
3. Immediately add the cell lysis buffer to the cells. The amount of
lysing solution needed depends on the cell density in the culture
vessel (for example, add 1.5–2 ml of lysis buffer to a 10 cm dish that
is ~80% confluent).
Note: It may be necessary to lyse the samples with different volumes of
cell lysis solution to obtain the specified protein concentration range.
4. Scrape the cells with a cell scraper, collect cell suspension into an
appropriately sized tube and gently rock for 20 min at 4°C.
5. Perform either of the following to remove insoluble cellular particulates:
n
Centrifuge the cell lysate solution at 4,500 x g for 20 min at 4°C,
and then filter the lysate using a 0.45 μm syringe filter
n
If the lysate volume is not adequate for filtration, centrifuge the lysate
at 15,000 x g for 10 min at 4°C using a benchtop microcentrifuge
6. Collect the filtered lysate or supernatant after centrifugation.
7. Measure protein concentration using Bio-Rad’s DC
™
protein assay
kit and if needed, adjust protein concentration with cell lysis buffer
containing PMSF and cell lysis factor QG.
8. The suggested working protein concentration range for Bio-Plex
®
cell signaling assays is 3–200 μg/ml (0.15–10 μg per assay well).
9. Store the aliquoted lysates at –70°C until ready to use.
9
Suspension Cells
1.
Collect cell suspension and pellet the cells by spinning at 1,000 x g for
5 min at 4°C.
2. Aspirate off cell culture medium completely.
3. Wash by resuspending the cells with ice-cold cell wash buffer
(bottle with the blue cap)
.
4. Centrifuge the cells at 1,000 x g for 5 min at 4ºC.
5. Completely remove the buffer.
6. Immediately add the proper volume of cell lysis buffer and gently rock
for 20 min at 4°C.
7. Remove insoluble cellular particulates as described in Adherent Cells
step 5 above
.
8. Follow Adherent Cells steps 6–9 above.
Tissue Samples
1. Cut the tissue into small pieces (~3 x 3 mm) for ease of handling and
blood removal. If necessary, wash the tissue with ice-cold cell wash
buffer (bottle with the blue cap) to completely remove all blood. Then
transfer the tissue to a 2 ml tissue grinder.
2. Add an adequate volume of cell lysis solution and grind the tissue
sample on ice using approximately 20 strokes.
Note: It may be necessary to lyse the samples with different volumes of
cell lysis solution to obtain the specified protein concentration range.
3. Transfer the ground tissue to a clean microcentrifuge tube and freeze
sample at –70°C. Freezing and thawing samples helps increase cell
lysis effects.
4. Thaw the sample and sonicate on ice (for example, with a Sonifier
450: Duty Cycle = 40, Output = 1, Pulse Sonicating = 18x).
5. Remove insoluble cellular matter as in Adherent Cells step 5 above.
6. Follow Adherent Cells steps 6–9 above.
10
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