Flow cytometric analysis of kinase signaling
cascades
Running title: FACS kinase analysis
Omar D. Perez1,2, and Garry P. Nolan1,2 *
1 The Baxter Laboratory for Genetic Pharmacology, Stanford University School of
Medicine, Stanford, CA 94305
2 Department of Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA 94305 \
Correspondence should be addressed to GPN: operez@stanford.edu,
Keywords: phospho-proteins, kinase activation, flow cytometry, proteomics, single-cell
Abstract
Flow
cytometry offers the capability to assess the heterogeneity of cellular subsets
that exist in complex populations, such as peripheral blood, based on
immunophenotypes. We describe methodologies to measure phospho-epitopes in
single cells as determinants of intracellular kinase activity. Multiparametric staining, using both surface
and intracellular stains, allows for the study of discrete biochemical events
in readily discernible lymphocyte subsets.
As such, the usage of multiparameter
flow cytometry to obtain proteomic information provides several major advantages
(1) the ability to perform multiparametric experiments to identify distinct
signaling profiles in defined lymphocyte populations, (2) simultaneous
correlation of multiple active kinases involved in signaling cascades, (3)
profiling of active kinase states to rapidly identify signaling signatures of
interest, and (4) provide biochemical
access to rare cell subsets such as those from clinically derived samples or
populations that comprise too few in numbers for conventional biochemical
analysis.
1. Introduction
Flow cytometry is routinely used for the
identification of cellular populations based on a surface phenotype and also
used for cellular based assays such as cytotoxicity, viability, and apoptosis,
among others. It is well understood that flow cytometry offers the capability
to assess the heterogeneity of cellular subsets that exist in complex
populations such as peripheral blood.
Current proteomic approaches, such as 2-dimensional SDS-PAGE and Mass-Spectroscopy
of protein post-translational modifications are extremely powerful and have
provided valuable insights into many intracellular activation processes.
However, as the cells are lysed, it is obvious that the readout of these
experiments is an average for protein activation states across the cell
population(s). Significant biology could be masked by such averaging, as there
is no provision for the collection of information on the distribution of
protein activation in individual cells within a population nor is there the
ability to retroactively identify the cellular populations that corresponded to
the detectable levels of active proteins. Therefore significant information on
human and mouse immune cell population variations that exist in both defined
cellular populations and across different cell subtypes is missed and cannot be
addressed by methodologies that require cell lysis for protein analysis.
Ultimately, protein activation signaling cascades must be measured in its most
biological context to be both relevant and free of artifact.
Thus, development of methodologies for intracellular
biochemical events, such as intracellular kinase activity measurements and
others in single cells will allow for a multiparametric approach to studying
discrete biochemical events of particular lymphocyte subsets existing in
complex heterogeneous populations. Multiparameter flow cytometric analysis
allows for small subpopulations – representing different cellular subsets,
differentiation or activation states – to be discerned using cell surface
markers. As such, the usage of single cell techniques to characterize signaling
events provides two major advantages (1) the ability to perform multiparametric
experiments to identify the distinct signaling junctures of particular
molecules in defined lymphocyte populations (2) obtain a global understanding
of the extent of signaling networks by correlating several active kinases
involved in signaling cascades simultaneously, at the single cell level 1.
1.1
Principle
At
present, the detection of active kinases is achieved by using phospho-specific
antibodies that differentiate between the phospho and non-phospho version of a
given protein. The generation of these highly specific antibodies (both
monoclonal and polyclonal) requires thorough testing to ensure not only
phospho-specificity but specificity against closely related proteins with
similar phosphorylation residues. Phospho-specific antibodies are conjugated
directly to fluorophores, evaluated for optimal fluorophore-to-protein ratios,
titrated for optimal concentrations, and tested under pre-defined stimulating
conditions. Occasionally, commercially available pharmacological inhibitors
exist that block specific signaling cascades and thereby provide a confirmation
of phospho-induction1, 2. However, for the majority of phospho-proteins,
these reagents do not exist and we provide alternative methods for determining
phospho-specificity (See below).
The technique for detecting
intracellular phospho-proteins with the greatest differential between induced
and uninduced, treated or untreated samples is a balance of several variables
that include the culture conditions, cellular manipulation, and specificity of
phospho-detecting reagent (antibody clone and fluorochrome ratio), cell
fixation, and cell permeabilization. In our laboratory, several protocols have
evolved that are designed to include protocols for phospho-detection alone,
phospho-detection plus surface markers, and phospho-detection plus surface
markers and other indicators of cellular function (i.e. apoptosis, cytokines).
In general, they differ in sequence of events and the methods of fixation and
permeabilization. Detergent based permeabilization methods such as saponin are
routinely used for intracellular cytokine detection. For instance with
phospho-protein analysis we found that for saponin based permeabilization it
was necessary to add combinations of phosphatase inhibitors to arrest
phosphatases activity prior to intracellular staining as fixation methods did
not abrogate all phosphatase activity within the cell nor in in vitro phosphatase activity assays (ODP and GPN unpublished results).
The saponin based permeabilization technique also maintained the integrity of
surface antigens, allowing us to readily discern between bright and low
expression as well as other parameters such as annexin-V staining1, 3. Alternatively, methanol permeabilization not only
inhibits all cellular activity within the cell, but it also denatures all
protein content, fully exposing intracellular epitopes for detection (cell
shapes are still intact because of the prior fixation step). Methanol also has
the benefit of allowing samples to be stored over time, a consideration for
clinical samples or samples in which analysis is not immediately possible. However, methanol permeabilization does
compromise detection of some surface antigens and makes population subgating
more difficult. Both techniques have their advantages and disadvantages. We
describe these in order of complexity and illustrate examples of each method.
It is not obvious apriori which technique is suited for particular kinases and
staining combinations. Therefore, the several protocols need to be evaluated by
the investigator and determined as appropriate for a particular analysis
setting.
Before carrying out the systems detailed below, the
novice reader is directed to a series of treatises on flow cytometry. First, basic elements of flow cytometry is
covered in various chapters throughout this methods series. Second, antibody conjugation and titration
protocols are available at http://herzenberg.stanford.edu/Protocols/default.htm.
Third, multi-color considerations, including cross-channel compensation for
multiparameter analysis are explained in the following references4,5,6. Once the
reader is comfortable in these different arenas it is possible to undertake
some of the more advanced sections below.
1.2 Cell
system and reagents—General Overview
Even
with individuals experienced in flow cytometric staining it is advised to start
with a simple single staining experiment before attempting multiple-active
kinase staining or phospho-proteins plus surface/other markers. Oftentimes
phospho-antibody specificities do not always exist pre-made for flow cytometry
for every circumstance that could be warranted. To overcome this, commercially available kits for most
fluorophore dyes have simplified the conjugation protocols for creating
antibody conjugates.
There are many considerations during the conjugation
of antibodies for intracellular flow cytometry. For instance, we find that an
acceptable range for fluorochrome-to-protein (FTP) ratios is more restricted
for intracellular phospho-flow then for other antibody conjugations. For
example, in Figure 1A, increasing the FTP ratio from 2.4 to 5.9, significantly
degraded the detection capability of a phospho-p44/42 antibody conjugated to
Alexa647, although this range is known to be acceptable for surface labeling.
Each fluorochrome therefore needs to be evaluated for optimal FTP ratios.
Overconjugation can result in interfering with the antigen recognizing
capability of the antibody and/or can result in intramolecular quenching by the
fluorophores. Therefore, antibody clones, concentrations, and FTP ratios need
to be evaluated for in-house conjugated phospho-antibody production.
For setting up staining for phospho-epitopes it is
best to start with an inexpensive and controllable resource. For this purpose cell lines are an
acceptable place to start. We have had experience with Jurkat, CH27, HL60,
U937, K562, NIH3T3 and web published reports have indicated that staining for
PC12, A431, MOLT-3, and human endothelial cells are also possible. Since most
cell line systems need to be optimized for maximal induction conditions
starting out with pre-defined conditions for phospho induction is
recommended. We have observed that
kinases and phosphorylations as measured by flow cytometry allows for sensitive
observations of activity given variations in stimulation and kinetics of
phosphorylation. It is necessary, for instance, to titre stimulation conditions
for a known amount of cells as cell density can affect the response seen to even
potent stimulators such as PMA and ionomycin (Figure 1B). Additionally, Figure
1C displays effects of temperature differences in the preparatory steps prior
to phospho-staining. The outlined protocols, below, require a complete fixation
and permeabilization of the cells in order for adequate detection of signaling
response. Time delays prior to a complete fixation can effect the signaling
responses observed (Figure 1C). Figure
2 shows examples of the differential between induced and uninduced states for several
phospho-specificities in U937 cells under optimally defined conditions. (See
Notes 1 for cell line considerations).
2.
Materials
There
are 4 different protocols and materials are divided per protocol. These
protocols are designed for several different flow cytometric applications.
2.1. For Method 1
1.
PBS 1X (phosphate buffered saline):Dissolve 1.44g Na2HP04,
0.24g KH2P04, 8g NaCl, 0.2g KCl in 850 ml distilled
water. Adjust pH to 7.4 with HCl and volume to 1 liter. Store at room
temperature.
2.
Fetal calf serum (standard cell culture FCS). Store
at 40C.
3.
Formaldehyde. Paraformaldehyde stocks of 4-36%
can be made or bought (we use 16% paraformaldehyde stocks from Electron
Microscopy Sciences (Catalog # 15710) (See Note 2 for preparation). Store at room temperature.
4. Staining media: 1X PBS + 4% FCS + 1mM sodium
azide. Store at 40C.
(See Note 3).
5. EDTA: Make 5 M concentrated stock and use
to make resuspension buffer (PBS + 1 mM EDTA). EDTA is used to avoid cell
clumps during flow cytometer
acquisition. Store at room temperature.
2.2 For Method 2
1.
Reagents above plus:
2.
Saponin
permeabilization buffer. Make concentrated 10% saponin: mix 10 g Saponin
(containing > 25% saponingen content, Sigma) with 100 mls PBS. Place
at 370C until saponin has dissolved with mild stirring. Sterile
filter (0.22 mL), and store at 40C.
(See Note 4).
3.
Saponin staining
buffer: Final saponin concentration for permeabilization should be no less than
0.1% per sample. A 0.2% solution is made to account for residual volume in
wells left after wash. (See Note 5). For 0.2% saponin solution, in 45 mls of
PBS, add 4 mls of FCS +1 ml of the 10% saponin stock. This will make the
saponin permeabilization buffer (0.2% saponin + 4% FCS stain buffer). Store at
40C.
2.3 For Method 3
1.
Reagents above plus:
2.
Phospho wash buffer: To
500 ml 1X PBS add 1mM b-glycerolphosphate, 1mM sodium
orthovanadate, 1 mg/ml microcystin (500 mg vials can be purchased through Calbiochem), 1mM
azide. This is the stock “phospho-stain wash” buffer and used as the diluent to
make up the other buffers. Store at 40C.
3.
Saponin staining
buffer. For 0.2% saponin solution, in 45 mls of phospho wash buffer, add 4 mls
of FCS +1 ml of the 10% saponin stock. This will make the saponin
permeabilization buffer (0.2% saponin + 4% FCS stain buffer). Store at 40C.
2.4. For Method 4
1.
Phospho wash buffer as
described above. This is the base buffer for all subsequent buffer
formulations.
2.
Extracellular staining
buffer: Phospho-wash buffer + 4% FCS+1mM azide, protease inhibitor cocktail
tablet (Boeringer Mannheim). Store at 40C.
3.
Fixation buffer: 1%
paraformaldehyde in phospho-wash buffer. Store at 40C.
4.
Permeabilization
buffer: 0.2% saponin + 4%FCS in phospho-wash buffer. Store at 40C.
3. Methods 1
Here we provide optimized protocols for the detection
of phospho-proteins and cytoplasmic proteins within intact cells. The
advantages of these protocols allow for the detection of signaling
intermediaries such as active kinases and intracellular proteins by flow
cytometry. The flow cytometric platform allows for a multiparametric assessment
of active kinases within immunophenotyped cells, among other parameters of
interest. The staining principles are
based on modified surface and intracellular staining procedures that were
optimized for detection of phospho-proteins. The procedures are best used for
suspension cells, although some success has been achieved with adherent
fibroblasts. Direct fluorophore conjugated antibodies are used in combinations
as they are best suited for multiparameter analyses, although indirect staining
is possible for 1 or 2 parameters. The procedures have been tested in a variety
of cell lines, primary mouse splenocytes, and primary human PBMC. Several
protocols are described and are suited for different applications.
3.1. Protocol 1 is designed for single phospho-staining.
3.1.1. Cell preparation
3.1.2. Fixation and permeabilization
3. Add
concentrated formaldehyde solution to cell cultures directly so that final
concentration of formaldehyde is 1-2% (i.e. if using 16% formaldehyde, add 100 mL per ml of sample). Swirl plate to homogenously
distribute fixative and return to 370C for 15 min.
4. Transfer
samples to FACs tubes by pipetting up and down to ensure complete cell removal
and place samples on ice. Activated
cells will tend to stick to the plastic, as will some others due to the
presence of the fixative. Pipetting up and down dislodges the majority of these
cells. Calculations of cell loss can be performed by counting cells before and
after. Check by microscope to determine that cells have been completely removed
as this is a frequent place where novices have experienced significant cell
loss.
5. Spin down
cells (1500 RPM, 40C, 5 min).
6.
Permeabilize cells by adding 1 ml of ice-cold methanol to cell tube
while it is being vortexed (medium speed). Addition of methanol is done at a
reasonable rate (i.e. 2-3 seconds for 1 ml) (See Note 7). Let sit on ice for 15
min. (See Note 8 for storage considerations).
7. Wash cells
3X with PBS (2-4 mls) to ensure removal of methanol. Washing with staining
media may result in FCS protein precipitates if methanol is still present and
is therefore to be avoided.
3.1.3. Staining.
8. Stimulated cells may be redistributed to
test several antibody stains originating from the same stimulated cell
population. Resuspend cells in staining media and aliquot 25-100 mL of cell sample. Cell number for antibody staining
should be titred so that 0.5-1 X106 cells can be allotted for each
stain in the 25-100 mL sample. The fewer the cells
per stain, the longer it can take for flow cytometry acquisition so it is
important to adjust accordingly. (See Note 9).
9. Add the primary antibody to the cell mixture
and incubate for 30 min (some reagents benefit from longer incubations i.e. 60
min). Typically, we make one uniform antibody staining cocktail for all samples
(i.e. if antibody is titred to 0.1 mL l/sample
and we have 10 samples, we make up N+1 samples in a final volume of 50 mL so that all the sample are stained with uniform
amounts of the reagent :
Reagent |
Amount |
X |
Samples |
Total |
antibody (concentration titred to
1X106 cells) |
0.1 mL |
|
11 |
1.1 mL |
Staining media: |
49.9 mL |
|
11 |
548.9 mL |
|
|
|
|
|
|
|
Total: |
|
550 mL = 50 mL /sample |
10. Wash cells 2X with 1 ml PBS and resuspend in 100 mL PBS/EDTA.
If the primary antibody was conjugated to a fluorophore, you may now
proceed to analyze the sample(s) on the flow cytometer. If using indirect staining
(i.e. secondary antibody) continue on to step 11.
11. Use fluorochrome-conjugated secondary at a pre-determined dilution
for 15 min (either manufacturer’s recommendation or 1 to 500-1000 is a starting
point). You want to achieve a low level of background staining using the
secondary alone control sample. This
staining should be similar to a directly-conjugated isotope control. It is
often required to titre the secondary for achieving a maximal signal differential.
12. Wash cells 2X in PBS,
resuspend in 100 mL PBS/EDTA and analyze by flow cytometer.
13. Flow cytometry voltages
are set to visualize the isotype control, or the secondary control at the
lowest possible range (i.e. below 101 log fluorescence). Uninduced
treated sample is then collected and induced sample is made relative to
uninduced. Sometimes culture conditions artefactually activate signaling
systems (thus the requirement for most cell lines to be serum starved). This, as noted raises the levels of some
kinases in the “uninduced” cell to a higher state of basal activation. (See Note 6 for additional staining
controls).
3.1.4. Staining for multiple kinases
simultaneously
The
ability to discern multiple activation states of proteins simultaneously within
the cell opens many opportunities to study signaling cascades, signaling
crosstalk, and the ability to monitor activation states over time. We have used
such approaches to gain insight in correlative bio-signatures and kinetics of stimuli
in time-referenced samples (Perez et al, manuscript submitted). In addition,
the ability to rapidly screen multiple targets simultaneously would be
advantageous in high –throughput drug screening, since these methods would
offer detection of kinase activities intracellularly (ensuring that
pharmaceutical regulators of key signaling component traversed cell membranes
or acted according to expectation) and target validation (verifying specificity
to desired protein and not other signaling molecules that may tap into the same
pathway).
To
assess multiple phospho-proteins simultaneously in the absence of surface
markers or other flow cytometric markers (i.e. apoptosis, DNA etc), the above
mentioned protocol is adequate. The second detecting antibody will be added to
the antibody cocktail (calculated appropriately as with the first example) and
the cocktail is applied to all samples. Increasing the number of intracellular
phospho-proteins detected will require the usage of directly-conjugated
antibodies since using monoclonal or polyclonal anti-phospho antibodies can
complicate staining considerations. The commercial availability of directly
conjugated anti-phospho specific antibodies is expected to meet the need of
most consumers; however certain reagents may still need in-house laboratory
generation.
Figure 3A demonstrates staining for kinases in 2 and 3 dimensions in human PBMC using IL-4 or IL-12 as stimulation. Figure 3B is demonstrating the activation of several MAPK pathways in primary cells that are cultured. This is an example of 1) the effects of culture conditions and background of phosphorylation levels, and 2) profiling of 3 MAPKs simultaneously.
3.1.5. Surface marker and intracellular
phospho-staining
In
complex cell populations it would be desired to distinguish cell subsets by
surface markers and then undertake intracellular phospho-protein staining in
1-step using either the saponin or methanol permeabilization techniques. To do
this requires first an evaluation of surface markers pre and post fixation and
permeabilization steps to assess maintenance of surface antigen detection. We
have used the saponin based technique (See protocol 3) in a 2-step staining
procedure to correlate phospho-profiles in immunologically defined cellular
subsets as complex as 11 parameters1. However, this procedure requires particular
attention to detail and requires the arresting of phosphatase activity by a
combination of phosphatase inhibitors and ice-cold buffers. If the methanol
permeabilization is performed, we have observed in some circumstances that
staining for surface antigens need to be evaluated. We have observed that as the epitopes on some surface proteins
are detectable only after extensive rehydration, others epitopes remain
compromised after methanol fixation, and yet others will be stripped from the
surface as they are maintained on the cells loosely and are removed by methanol
fixation. As before, it is necessary to titrate the antibodies to surface
epitopes. (See Note 10).
In a series of sequential surface staining
experiments, in which 200+ surface antibodies on all colors were profiled for
the ability to stain in paraformaldehyde, saponin, and methanol pre-fixation,
post-fixation, pre-permeabilization, post-permeabilization, as well as
determining paraformaldehyde’s, saponin’s, and methanol’s affect on
fluorochrome intensity to a panel of protein and in-organic dyes, it was
determined that in general, the best staining combination was to be performed
post fixation and post permeabilization however it wasn’t predictable on
antibody (clone or antigen) or
fluorochrome which ones would work the best (ODP and GPN data not
shown). The majority of the dyes were not affected by paraformaldehyde and or
saponin treatments, but a majority of the antibody reagents were compromised by
the methanol treatment.
For example, Figure 4 displays surface detection of
typical T cell markers, CD4, CD3, CD62L, and CD8 under various pre- and post
fixation and permeabilization conditions. These antibodies survived both the
fixation and permeabilization conditions. Staining post fixation and
permeabilization (saponin), it is noted that forward scatter size has been
reduced, and the additional population contained within the lymphocyte gating
is reflected in the intermediate populations showing up in the fluorescent
parameters that are typically excluded (Figure 4 column IV). Methanol
permeabilization in this example, increased CD3 staining background and
abrogated CD8 surface staining (Figure 4, column V). Therefore, for these
specific monoclonal antibodies the permeabilization/staining conditions that
work best are found in column III
3.2. Method
2
Protocol
2: Surface + intracellular staining (methanol rehydration protocol).
An example of this protocol’s staining is shown in Figure 5A, where human PBMC was treated with various cytokines, stained for both surface (CD56 and CD11b— markers for NK cells and MAC-1 expression) and intracellular markers (phospho-STAT6) simultaneously and then gated for lymphocyte subset signaling differences. The results show that CD56+ populations phosphorylate STAT6 upon stimulation with IL-4 and IL-12. CD56+ populations with higher levels of CD11b in general had elevated levels of phospho-STAT6. CD56-CD11bhigh populations did not display changes in phospho-STAT6 to stimulations tested.
3.2.1 Cell
preparation and cell fixation/permeabilization
These
procedures are the same as those outlined in protocol 1. After step 7 in
protocol 1, resuspend the cells in 500 mL of staining
media (4% FCS in PBS) for 1 hour. The extensive washing and rehydration step
increases detection of many epitopes for human surface antigens. For reasons we do not understand, at
present, we do not observe as many difficulties with murine surface epitopes.
3.2.2 Staining
8. Antibody
cocktails are made up in staining media as was exemplified in protocol 1.
Multi-color work requires staining with individual antibody-fluorophore
conjugates to be used as compensation controls9. In the antibody cocktails, 1/5 of the final volume
of the antibody cocktail should be staining media containing 4% FCS. If higher
amounts of diluted antibodies are used, the final volume of the antibody
cocktail can be increased to 100 mL with
staining media. An example of a 4 color setup is provided below.
Total
volume = 50 ml/1X106 cells
For
“X” amount of samples, make up sufficient reagents for “X+1”
Reagent |
Vol per sample |
# samples |
Total volume reqd |
Ab-FITC |
5 mL |
5 |
25 mL |
Ab-PE |
6 mL |
5 |
30 mL |
Ab-PercP |
10 mL |
5 |
50 mL |
Ab-APC |
2 mL |
5 |
10 mL |
Staining Media |
27 mL |
5 |
135 mL |
|
|
|
|
Total |
50 mL |
|
250 mL |
Since
the final volume per sample is 50 mL, it is
adjusted using staining media. A minimum of 10-15 mL of staining media is needed to ensure blocking
agents are included in the stain, thus if diluted antibodies are used, it might
be necessary to scale up to a 100 mL staining
volume.
10.
Antibody stains are done for 1 hour at 40C on ice (covered to
protect from light).
11. Cells are washed 3x in PBS and
resuspended in 100 mL PBS/EDTA and analyzed by flow
cytometry.
3.2.3. Single step surface marker
staining for antigens requiring discrimination between bright and dim and
med/high
As
noted with methanol permeabilization, there can be a loss of staining for certain surface epitopes as well as a loss
of distinctive levels of expression (such as with CD11a, CD45RA, CD62L) and
between medium and high (such as with CD8 populations) tend to collapse or strongly overlap (Figure 4, column V). In
addition, the SS (side scatter) and FS (forward scatter) properties are not
always maintained with methanol or saponin permeabilization (Figure 4 column IV
and V) in 1-step staining procedures.
Since some PBL cell populations are readily discernable by size (such as
monocytes and lymphocytes) this can cause difficulties during analysis. Due to
such considerations we have applied saponin based techniques for fine cellular
subset characterization. (See Note 9).
3.3. Method 3
Protocol 3: Surface + intracellular staining (saponin
protocol)
An
example of this protocol is shown in Figure 5B where human PBMC was treated
with various cytokines, stained for both surface (CD16 and CD19) and
intracellular markers (phospho-STAT6) simultaneously and displayed for
lymphocyte subset signaling differences. The results show that CD19+ cells
phosphorylated STAT-6 upon stimulation of IL-4 and IL-12. Stimulation with TNFa slightly induced phosphorylation in some CD19+
cells, whereas IFNg and GMCSF did not induce
phosphorylation of STAT6. CD16+ cells did not display significant changes to
STAT-6 phosphorylation upon stimulations tested. Aspects of these findings are
further supported by various studies10; 11.
3.3.1. Cell
preparation.
Cells are prepared as described in protocol 1.
3.3.2.
Fixation and permeabilization
Cells
are fixed as described in protocol 1.
12. Cells are permeabilized in 200 mL saponin permeabilizing buffer for 15 min on ice.
Cells are then pelletted (1500 RPMI, 40C, 5 min). (See Note 5 for
alternative permeabilization considerations).
3.3.3. Staining
13. Cells are stained in antibody cocktails as described
for protocol 2 except the staining media used is the saponin based buffer. ****
Very important that the saponin based buffer is used for antibody cocktail, as
saponin permeabilization is reversible and, if using the standard staining
media, the antibodies for intracellular staining will not gain access to the
appropriate compartments.
14. Cells are stained for 1 hour at 40C
and protected from light.
15. Cells are
washed 3X in PBS and resuspended in 100 mL PBS/EDTA
and analyzed by flow cytometry.
3.3.4. Surface marker, intracellular
phospho-staining, and staining for other intracellular proteins or flow
parameters (i.e., cytokines, annexin-V, non-phospho proteins).
With
the advent of instrumentation that is capable of processing up to 15 simultaneous
parameters and with an appreciable desire to undertake biochemical analyses in
rare cell subsets that require many surface markers to identify, the
application of this methodology can provide correlative information on cellular
subsets that are not possible to analyze by conventional biochemical
approaches. It is also important to obtain as much information from a given
sample if such a sample might be limited in its availability (i.e. diseased
patient samples). In addition, combining phospho-profiling with detection of
other parameters such as intracellular cytokine production and cellular states
(such as cell cycle or apoptosis) can provide correlative information of
surface phenotype, signal transduction, and effector function in a single
experiment (Perez et al submitted, 3).
At present, we have been successful
in combining intracellular phospho and cytokine detection, as well as
intracellular phospho and the annexin-V apoptotic marker using the saponin
based buffers as outlined in protocol 4. Alternatively protocol 3 can be used
for these efforts, though our previous work has utilized the protocol outlined
below. We have not currently evaluated if the methanol protocols are adequate
for intracellular cytokine detection or other markers such as annexin-V.
3.4. Method 4
Protocol
4: Combining intracellular phospho-protein and cytokine staining, and surface
markers (See Note 11 for attention to particular steps in this protocol).
These procedures are best used on freshly
isolated PBMC. Isolation of PBMC is done by Ficoll-plaque density
centrifugation, PBMC are either used directly or enriched for particular
populations of interest by cell sorting (magnetic activated cell sorting or
fluorescence-activated cell sorting). Cells are then stimulated and processed
for staining. (See Note 12 for live/dead cell discrimination for intracellular
staining).
3.4.1. Cell preparation
3.4.2. Surface
Staining
3.4.3. Fixation and Permeabilization
3.4.4. Intracellular Stain
3.5 Summary
We
describe here several protocols to assess signaling in cells by intracellular
staining of phospho-epitopes (See Note 13 for specificity testing). The
considerations for staining will inherently vary in the application desired
(See Note 14 for adherent cell considerations). Still, the ability to
biochemically differentiate signaling responses in lymphocyte subsets by
multiparameter assessment will be a powerful and exciting opportunity to
investigate samples in which conventional biochemical techniques are not
suitable. This methodology is readily applicable to clinical samples or cells
from diseased blood to monitor signaling changes upon disease onset and
hopefully allow correlations of intracellular signaling events with clinical
parameters as a diagnostic indicator of disease progression. The development of
analytical software capable of processing multivariate data to obtain both
statistical and relevant information from flow cytometric data will greatly aid
this effort. For example in Figure 6, FACS based cluster analysis can identify
populations of interest from multparameter experiments, without prior
subjectivity of gating, and is a step towards automation of data analysis.
Furthermore, the assessment of multiple targets simultaneously offers
advantages in both biochemical assessment of signaling networks and the
potential for high throughput FACS based screens for specific modulatory
agents.
4. Notes
1. Considerations for using cell lines.
It is well understood that cell lines do not always functionally represent
primary cells and that differences exist between mouse and human systems. It is
also understood that model systems are dependent on the experimental
conditions. For most cell lines adapted to culture, we have found it is often
necessary to serum starve the cells for a period prior to subsequent stimulus
and phospho-detection. This time period is variable as 6-12 hours is typically
required for most cells, and prolonged periods can result in stress-induced
phosphorylation events. These conditions need to be determined prior to flow
cytometric detection as serum starvation may not be suitable for all
applications. Cell density and contamination (bacteria, yeast, and antibiotic
salvaged cultures) will affect the signaling responses of the cells. For
suspension cells, we recommend a cell density of 1-5 X105 cell/ml.
Too high of a cell density can changes the signaling properties of most cells
(in particular Jurkat 12). Frozen cells have compromised signaling and should
be allowed time to recover from freezing.
2. Paraformaldehyde
preparation. Paraformaldehyde is
toxic and volatile. Avoid breathing either fumes from dissolved
paraformaldehyde or powder. Use a fume hood as necessary. Mix a required amount of paraformaldehyde (e.g. 4% is
4g/100ml (w/v)) to 2/3 final volume in ddH2O. Heat to 600C while
stirring in a fume hood (monitor temperature with thermometer and avoid boiling
because it can volatilize and pose a serious hazard for respiratory and mucus
membranes and is therefore especially hazardous for contact lens wearers). Add
50-100 ml of 2N NaOH
to clear the solution required for appropriate solubilization). Remove flask from heat and add 1/3 volume
3x PBS. Let cool and adjust to pH 7.2 with HCL. Filter and store at room
temperature.
3. Blocking agents. Staining media contains fetal calf serum as a blocking
agent. FCS is generally a good blocking agent for most specificities. However,
detection of some specificities may be enhanced by using bovine serum albumin
instead as it is typically used in phospho-western blotting. Milk is not
recommended as it contains phospho-proteins that can result in higher
background.
4. Saponin composition. Saponin is a glycoside derived from plants
such as the Quillaja bark or produced
synthetically. Saponins are natural surfactants and comprise of several
different, but related molecules, triterpenoid structures that consist of
aglycone (saponingen) structure with glycosyl moieties. The purity and chemical
composition of commercially sold preparations will vary. We have observed that
the final concentrations of the saponin content based buffers (w/v) are best
using saponin containing > 25% saponingen content. The saponin buffer
will be yellow in color, and is to be stored sterile at 40C.
5. Considerations for saponin
based permeabilization.
Saponin concentrations for efficient permeabilization range from 0.1%-0.5% Too high concentrations of saponin start to
destroy membranes and results in compromised stains. Final saponin
concentration for permeabilization should be no less than 0.1% per sample.
Often a 0.2% solution is made to account for residual volume in wells left
after wash. The final staining concentration range is 0.1% - 0.5%. Saponin permeabilizes
membranes by solvating sterol molecules (i.e. cholesterol molecules). The
permeabilization is reversible and therefore, antibody cocktails need to be
made in the saponin based buffers for entry into the cell. Saponin
permeabilization is sufficient for various nuclear, mitochondrial, endoplasmic
reticulum, and granule located proteins as we have confirmed staining patterns
by confocal microscopy. Saponin permeabilization may not be suited for all
phospho-epitopes. This is believed to be due to the in-accessibility of some
epitopes in protein-protein interactions. For these reasons, comparing the same
induction conditions using the methanol procedures may be necessary.
Alternatively, Triton-X-100 at 0.5% can be used to permeabilize cells (5 min, then
washed out prior to antibody cocktail stains) as a way to permeabilize cells
and retain surface antigen integrity. Also, Pharmingen’s Cytofix/cytoperm
buffer works well in this protocol, provided that the subsequent staining step
is done in the permeabilization buffer (using plain staining media was not as
effective). The combination fixation/permeabilization buffers greatly benefited
from a second permeabilization step.
6. FC receptor blocking. When staining in PBMC, often times, FC receptor
bearing cells bind some antibody isotopes and therefore one must add blocking
agents such as non-specific mouse IgG or corresponding to the isotype
recognized by the FC. This is of particular importance if using secondary
staining techniques, where directly conjugated Fab fragments might be
considered.
7.
Efficient permeabilization using
methanol. Adding the entire 1 ml of methanol rapidly to the cells prior to
their resuspension by vortexing can result in 1) inefficient permeabilization
in some cell types and/or 2) cells sticking to the plastic and resultant cell
loss.
8.
Storage of fixed and permeabilized
samples. Cells can be stored at -200C in methanol or be
processed directly afterwards. Samples in our hands have been stored for short
term (several days to 2-3 weeks). We are currently evaluating longer term
storage conditions.
9.
Antibody titrations. Appropriate
titration of antibodies is critical for optimal detection of phospho-epitopes--
in addition to cost savings in antibody usage. We typically titre all our
surface and intracellular antibodies to a standard of 1 X106 cells.
For intracellular phospho-stains, we use defined stimulation conditions and
obtain the concentrations that allow for the maximum differential to be
detected using the background of isotype controls or unstimulated controls as a
measure against non-specific staining events. We also used a fixed number of
cells to titre stimulations in order to obtain fixed concentrations of
stimulating agent (i.e. cytokines). Cell density affects both the efficiency of
antibody detection, stimulation conditions, as well as reproducibility in these
protocols Note that direct comparison of methanol and saponin methods present
differences in the induction levels detected and the titration of the
intracellular antibodies required. Often, titres of intracellular antibodies
for saponin methods are higher than that for methanol permeabilization.
Typically, we observe that intracellular phospho-antibodies stain at ~0.25-1 mg / 1X106 cells for saponin based
protocols, and 0.05-0.25 mg/ 1X106 cells for
methanol protocols.
10.
Considerations for 1-step staining
protocols. This also requires a thorough washing of the cells since some
surface antibodies may react non-specifically with intracellular epitopes, as
well as detect intracellular agents that are contained in vesicles waiting to
be surface expressed upon stimulation.
11.
Inhibition of intracellular phosphatase
activity. Phosphatase inhibitors
were critical for the 2-step saponin based procedure as was ice-chilled buffers
and refrigerated centrifuge spins. The fixing/permeabilization techniques did
not completely abolish all phosphatases activity, and for these reasons the
phosphatase inhibitors, and keeping everything on ice or at 40C, is
essential. The phosphatase inhibitors we have chosen were selected on the basis
that these inhibit the majority of known or abundant phosphatases. There are
essentially 4 classes of phosphatases: alkaline phosphatases, acid
phosphatases, protein tyrosine phosphatases, and serine/threonine phosphatases
(these categorizations are not exhaustive). Depending on the kinase being
detected it is advised to choose the phosphatase cocktail best suited for the
application. Combine them if detecting different kinds of phosphorylation and
take into consideration any special properties of a kinase of choice (i.e.
kinetics of activation, rate of dephosphorylation, etc). We also added protease inhibitors to our
buffers, 1 mM azide if the buffer is to be stored for some time (2-4 weeks).
EDTA is not recommended in the subsequent buffers since divalent ions are
needed for some antibody detection (and other agents such as annexin-V).
Buffers are stored at 40C and are stable for 1 week. Protease
cocktail tablets, commercially available through Boeringer-Mannheim, were added
to buffers on a per usage basis.
Protocol 3 and 4 utilize phosphatase inhibitors to
aid in the detection. The addition of b-glycerolphosphate,
sodium orthovanadate and microcystin are the minimum combinations of
phosphatases inhibitors needed to inhibit the majority of tyrosine and
serine/threonine phosphatases. There are other reagents that inhibit
phosphatases that may be considered for enhanced detection (i.e. calyculin A,
okadaic acid, sodium fluoride). These inhibitors are not as necessary with the
methanol permeabilization since methanol permeabilization abrogates the
majority of phosphatase and enzymatic activity.
12.
Live/dead cell discrimination. When
performing intracellular stains, it is important to distinguish between live
and dead cells by methods other than forward and side scatter gating since
these parameters can and do change upon the fixation/permeabilization
conditions. Dead cells often bind a
large number of antibodies non-specifically.
Cell viability is routinely performed by membrane exclusion dyes such as
propidium iodide (PI). If a cell’s membrane has been compromised, they stain
with PI and are so labeled “dead”. PI
is often read in the 670 nm channel of a 488 nm laser excitation. PI is useful as a live-dead discriminator
when one is only undertaking surface staining alone, but is not adequate for
intracellular staining where the permeabilization conditions can will
inadvertently label all cells as dead. For this reason, it is critical to use
the compound such as EMA (ethidium monoazide). EMA is an ethidium bromide
analogue that is excluded by intact cellular membranes, but can enter dead
cells and, in the presence of light, forms a covalent adduct with DNA.
Therefore, cells in which the membranes have been compromised prior to
permeabilization or fixation are permanently labeled as ‘dead’ with EMA.
Subsequent permeabilization does not affect this compound, making it a superior
discriminator of live/dead cells when intracellular staining is performed. It
is absolutely critical when working with cell populations that comprise less
than 1-5% of the total cell population (i.e. lymphocyte subsets within PBMC) as
dead cells bind antibodies non-specifically13. EMA is
equivalent in fluorescence to PI and is commercially available through
Molecular Probes. If using EMA, final conc. = 5 mg/ml
(make as stock of 5 mg/ml, 1000X then use 5 mL
per 100 mL stain of EMA). EMA is either added in the first
wash or with surface antibody cocktail. Cells are stained and maintained in the
dark. After surface stains are washed, the tubes are pulsed with light (or held
up a fluorescence light bulb for 30 seconds), then the rest of the staining
procedure can be carried out as outlined.
13.
Testing for phospho-specificity in the
absence of specific pharmacological agents. It is necessary to confirm that
the increased staining for phospho-epitopes observed is due to the biochemical
reality of the cognate phosphorylation event. Pharmacological agents that will
inhibit the phosphorylation of the protein of interest are not always available
to allow such verification. In such examples, it is often necessary to use one
of four alternatives: (1) out-compete phospho-antibody with phospho-peptide,
(2) out-compete phospho-antibody binding with phenyl phosphate solution, (3)
test for phospho-specificity upon phosphatase treatment of cellular samples
after an activation condition has been established, or 4) obtain or generate
kinase deficient, site-directed mutagenized kinase expressing cells (i.e.
tyrosine sites mutagenized to phenylalanine), or knockout cells to test
detection of phosho-specificity First,
conditions need to be established that will render phosphorylation of the
protein of interest above unstimulated controls, and verified by traditional
immunoblot analysis. Second, these same conditions must be tested on a flow
platform, and be above the background of the unstimulated cells. Post
activation, a sample of cells can be fixed, permeabilized, and treated with (1)
1 mg of phospho-peptide, (2) resuspended in 10 mM
phenylphosphate solution or (3) treated with
phosphatases, such as lambda phosphatase (1-5 Units, 30 min, 370C)
in the same intracellular staining conditions established. Subsequent washing
and intracellular staining with the phospho-specific antibody should be
significantly decreased compared to activated and stained cells. Titration experiments can readily be applied
with both the phenylphosphate and phospho-peptide. Alternatively, incubating
permeabilized cellular samples with phospho-tyrosine, phospho-serine, and
phospho-threonine solutions (both free chemicals and conjugated to BSA) can be
used to raise the background to ensure titrations are appropriate as these
treatments would increase the overall background. We are currently determining if other approaches, such as RNAi
can be more broadly applied to verify target staining observed is real and not
artefactual.
14.
Adherent cells and tissue samples. We
have optimized the protocols for suspension cells, although have had success
with adherent fibroblast such as NIH3T3 for intracellular phospho-staining. We
have utilized two approaches for adherent cells, and at present haven’t
extensively compared the methods directly. Adherent cells can be removed from
the plate using a PBS/EDTA solution, and not trypsinized or scraped off (to do
so may destroy cell integrity). For NIH3T3 cells, cell permeable phosphatase
inhibitors in the PBS/EDTA buffer greatly enhanced phospho-detection. We note
that washing and centrifugation steps can affect signaling systems within cells
and should be determined upon an experimental basis if considered a concern.
Adherent cells can also be fixed first and then “gently” scraped off into FACS
tubes for permeabilization, however, a significant proportion of the cells will
be fixed onto the plastic or can be destroyed in the scraping. This is a
concern since most activated cells, the cells of interest are the ones that
adhere more strongly.
The potential to analyze phospho-signatures from
tissues and biopsy sections is a promising application of this methodology. We
have not optimized these protocols for cell samples taken from tissues since
considerable cell manipulation is required to extract single-cell suspensions.
Although tissues taken from mice have been demonstrated to be amenable to these
procedures14; 15; 16. We hope that in the future we will adapt these
techniques to be able to profile phospho-signatures of cells derived from human
tissue sections or tumor masses.
Acknowledgments
We acknowledge support and helpful discussion from BD Biosciences-PharMingen and the resources of the Stanford FACS facility. We are indebted to Leonard and Leonore Herzenberg for their consistent support in pursuit of these endeavors. We thank the members of the Nolan laboratory for many helpful discussions, including Jonathan Irish, Matt Hale, and Peter Krutzik. ODP was supported as a Bristol-Meyer Squibb -- Irvington-Institute Fellow and the National Heart, Lung, and Blood Institute contract N01-HV-28183I. GPN was supported by NIH grants P01-AI39646, AR44565, AI35304, N01-AR-6-2227, A1/GF41520-01, NHLBI contract N01-HV-28183, and the Juvenile Diabetes Foundation.
5.
References
1. Perez, O. D. & Nolan, G. P. (2002). Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20, 155-162.
2. Chow, S., Patel, H. & Hedley, D. W. (2001). Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 46, 72-8.
3. Perez, O. D., Kinoshita, S., Hitoshi, Y., Payan, D. G., Kitamura, T., Nolan, G. P. & Lorens, J. B. (2002). Activation of the PKB/AKT Pathway by ICAM-2. Immunity 16, 51-65.
4. Roederer, M., De Rosa, S., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D. & Herzenberg, L. (1997). 8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29, 328-39.
5. Baumgarth, N. & Roederer, M. (2000). A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 243, 77-97.
6. De Rosa, S. C., Herzenberg, L. A. & Roederer, M. (2001). 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat Med 7, 245-8.
7. Shan, X., Czar, M. J., Bunnell, S. C., Liu, P., Liu, Y., Schwartzberg, P. L. & Wange, R. L. (2000). Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 20, 6945-57.
8. Freeburn, R. W., Wright, K. L., Burgess, S. J., Astoul, E., Cantrell, D. A. & Ward, S. G. (2002). Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors. J Immunol 169, 5441-50.
9. Roederer, M. (2001). Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45, 194-205.
10. Rudge, E. U., Cutler, A. J., Pritchard, N. R. & Smith, K. G. (2002). Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression. J Exp Med 195, 1079-85.
11. Morris, S. C., Dragula, N. L. & Finkelman, F. D. (2002). IL-4 promotes Stat6-dependent survival of autoreactive B cells in vivo without inducing autoantibody production. J Immunol 169, 1696-704.
12. Fiering, S., Northrop, J. P., Nolan, G. P., Mattila, P. S., Crabtree, G. R. & Herzenberg, L. A. (1990). Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev 4, 1823-34.
13. Herzenberg, L. A., Parks, D., Sahaf, B., Perez, O. & Roederer, M. (2002). The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem 48, 1819-27.
14. Zell, T., Khoruts, A., Ingulli, E., Bonnevier, J. L., Mueller, D. L. & Jenkins, M. K. (2001). Single-cell analysis of signal transduction in CD4 T cells stimulated by antigen in vivo. Proc Natl Acad Sci U S A 98, 10805-10.
15. Zell, T. & Jenkins, M. K. (2002). Flow cytometric analysis of T cell receptor signal transduction. Sci STKE 2002, PL5.
16. Kaech, S. M., Hemby, S., Kersh, E. & Ahmed, R. (2002). Molecular and functional profiling of memory CD8 T cell differentiation. Cell 111, 837-51.
Figure legends
|
Figure 1. Effect of FTP ratio,cell density, and
time of fixation on signaling detected by flow cytometry. A)
1X106 serum starved (12 hrs) Jurkat cells were stimulated with
PMA/IO (500 ng/ml, 15 min). Cells were fixed, permeabilzed and stained (as per
protocol 1) with anti-phospho-p44/42(T202/Y204) (clone 20a) conjugated to
varying ratios of Alexa-647 as indicated in figure. 0.125 mg of antibody was used for all stains. Geometric
mean values were computed as a ratio of stimulated to unstimulated, and
plotted as a function of FTP ratio in graph.
B) Jurkat cells were stimulated with PMA/IO (500 ng/ml, 15 min) at
indicated densities and prepared as described above. C) Unstimulated Jurkat
cells were either fixed after washing or directly fixed prior to washing.
Cells were washed at 370C or 40C (including
temperatures of buffers and centrifugation step) and permeabilized using
either methanol or saponin based protocols. Phospho-p44/42-AX647 (clone 20a)
was used as antibody stain. |
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|
Figure 2. Display of several phospho-specificities.
1 X106 serum starved (12 hrs) U937 cells were
stimulated with either anisomycin (20 mM), PMA/IO
(500 ng/ml), PHA (50 mg/ml),
IFNg (200 ng/ml), IL-4 (200 ng/ml),
or GMCSF (200 ng/ml) for 15 min. Cells were fixed, permeabilized and stained
(as per protocol 1) with phospho-p38(T180/Y182)-AX647 (clone 36),
phospho-p44/42(T202/Y204)-AX647 (clone 20a), PY20-PE,
phospho-STAT1(Y701)-AX488 (clone 14), phospho-STAT6(Y641)-AX488 (clone 18),
phospho-STAT5(Y694)-AX488 (clone 47). Antibodies were used at 0.125 mg. |
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|
Figure 3. Phospho-staining in 2 and 3 dimensions. A) 1X106
CD4+ naïve T cells (purified by negative isolation) were treated with IL-4 or
IL-12 (200 ng/ml) for 12 hours. Cells were fixed, permeabilized, and stained (as
per protocol 2) and stained with phospho-STAT6(Y641)-AX488 (clone 18) and
phospho-STAT1(Y701)-AX647 (clone 4a). Antibodies were used at 0.25 mg. B) Three MAPK kinase signaling responses
simulataneously. Human PBMC depleted for adherent cells were cultured in
either 10% autologous human sera or 10% FCS (Hyclone). Cells were stained for
phospho-p44/42(T202/Y204)-AX488 (clone 20a), phospho-p38(T180/Y182)-PE (clone
36), and phospho-JNK(T183/Y185)-AX647 (clone 41) as per protocol 1.
Antibodies were used at 0.125 mg
(p-p44/42) and 0.25 mg (p-p38
and p-JNK). |
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|
Figure 4: Sequential staining of surface antigens upon
fixative and permeabilization treatments. 1X106 PBMC
were either surface stained (column I), surface stained then fixed in 1%
paraformaldehyde (column II), surface stained, fixed in 1% paraformaldehyde,
then permeabilized by 0.2% saponin (column III), fixed, permeabilized (0.2%
saponin), then surface stained (column IV), or fixed, permeabilized
(methanol), then surface stained (column V). Cells were stained with
CD62L-FITC (clone DREG 56), CD4-PE (clone RPA-T4), CD8-PercpCy5.5 (clone
SK1), and CD3-APC (clone UCHT1). Top row displays forward (FSC) and side
scatter (SSC) profiles, and lymphocyte gate used for display of subsequent
rows. Figure displays consequences of antigen staining towards fixation and
permeabilization conditions for intracellular staining. Post
permeabilization, oftentimes, reduction in the forward scatter is observed,
and the appearance of these populations is illustrated by the intermediate
populations appearing in the surface marker staining that are typically
excluded by lymphocyte gating alone. Column VI displays the ungated surface
stains alone. |
|
|
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|
Figure 5. Multiparameter staining: surface+intracellular
staining A) 1X106
PBMC (non-depleted) were
treated with indicated cytokine (200 ng/ml, 15 min) and stained for
CD16-cychrome (clone 3G8), CD19-PE (clone HIB19), and
phospho-STAT6(Y641)-AX647 (clone 18)
(as per protocol 3). Cells were gated for either CD16 or CD19 and
displayed for phospho-STAT6. B) Cells
were prepared as described above and stained with CD56-PE (clone B159),
CD11B-cychrome (clone ICRF44) and phospho-STAT6(Y641)-AX647 (clone 18) (as
per protocol 4). |
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|
Figure 6. Multivariate analysis of complex
populations 1X106 PBMC (non-depleted) were left untreated or treated with
indicated TNFa (200
ng/ml, 15 min) and stained for CD14-FITC (clone MfP9), CD19-Cychrome (clone HIB19),
phospho-STAT3(Y705)-PE (clone 4), and phospho-STAT5(Y694)-AX647 (clone 47) (as per protocol 4).
Multivariate analysis was performed using clustering algorithms developed by
Dr. Mario Roederer in Flowjo 4.2 |
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