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



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

  1. For cell cultures, plate 1 X106 cells per ml in standard tissue culture plates (6-, 12-, or 24,-well plates). Cell lines typically must often be serum starved for up to 12 hours (times may vary). For example, Jurkat cells, although useful as a model for T cell studies, have genetic defects in the PTEN and SHP-1 phosphatases7,8, consequences of which allow for sustained signaling through the AKT and PI3-Kinase effector pathways. Such genetic mutations enable Jurkat cells to proliferate in culture in comparison to naïve T cells, which require exogenous stimulation to proliferate. Often, cells grown in high concentrations of a non-native serum component such as calf serum require high concentrations of growth factors to supply needed stimulants that the cells no longer obtain from their native milieu.  Thus, they are ‘adapted’ for growth in a non-native environment in which they are often hyperstimulated.  Many signaling systems are not, under such conditions, at a basal state and their background activation is readily apparent.  1X106 cells are used during initial titration of antibodies. It is important to understand that titration of antibodies per cell number is a critical prerequisite to obtaining optimal signal to noise. This is due to the fact that the best antibody concentration and effectiveness at binding to targets within the cell is a function of the number of target phospho-epitopes to be bound per cell, the number of cells, appropriate concentration of non-specific binding blocking agents, and the background binding events that can occur Once optimal conditions have been determined the cell number and antibody amounts can be scaled accordingly. If multiple different stains are to be undertaken with from the same sample, it is necessary to scale up the cell number so that after the fixation/permeabilization the sample can be split up into several assay tubes for individualized staining and treatment.
  2. Add cell stimulation at desired concentration. Make provisions for control stains in addition to unstimulated/stimulated samples (i.e. isotype control if available, or secondary alone stain if performing indirect stains) (See Note 6 for additional controls). Return to 37 0C for 15 min.


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 :








antibody (concentration titred to 1X106 cells)

0.1 mL        



1.1  mL                           

Staining media:

49.9  mL       



548.9  mL










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.

  1. Antibodies used for either surface or intracellular staining can be used in cocktails once an optimal concentration has been determined. A typical example of an antibody cocktail protocol is presented below:


            Total volume = 50 ml/1X106 cells

           For “X” amount of samples, make up sufficient reagents for “X+1”



Vol per sample

# samples

Total volume reqd


5 mL


25 mL


6 mL


30 mL


10 mL


50 mL


2 mL


10 mL

Staining Media

27 mL


135 mL






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

  1. PBMC (or purified cells) are dispensed in 96-well U-bottom plates at 0.5-1X106 cells per well in 100 mL of media.
  2. Cells are stimulated for desired stimulus, and length of time at 370C.
  3. Set aside controls: (1) Single color controls for all colors used (both positive and negative). (2) Controls for phospho-proteins (i.e. stimulated vs. non-stimulated) (3) Unlabeled control for autofluorescence (4) Intracellular isotype controls for background staining.
  4. Cells are harvested, by adding 100 mL of phospho-wash buffer, and centrifuged (1500 RPM, 40C, 5 min), plate is flicked, and immediately resuspended in ice-cold extracellular staining buffer (50 mL for 1-2 x 106 cell) and placed at 40C.


3.4.2. Surface Staining

  1. Samples are Incubated with surface cocktail (50 mL in extracellular staining) for 15 min on ice in the dark.
  2. Add 150 mL phospho-wash buffer and spin (1500 RPM, 40C). Wash 1x with 200 mL.


3.4.3. Fixation and Permeabilization

  1. Fix using 100 mL fixation buffer on ice for 30 min, in the dark. Final concentration should be between 1-2% if using a stock solution.
  2. Add 100 mL wash buffer, and pellet the cells. Wash 1X, 200 mL, pellet cells
  3. Permeabilize with 200 mL permeabilization buffer, pipette up and down 4-5 times. Incubate for 15 min , 40C, in the dark.
  4. Add 100 mL wash buffer, spin down cells, and flick plate.


3.4.4. Intracellular Stain

  1. Resuspend in 50 mL intracellular stain cocktail (made up in permeabilization buffer), at least 30 min on ice in dark (usually sufficient but longer time incubations (1 hr) and incubating at room temp can increase some staining. Add 150 mL, of permeabilization buffer, spin down.
  2. Wash 1-2X in 200 mL permeabilization buffer (two washes is usually sufficient, but more washes may decrease background)
  3. Resuspend in PBS/EDTA (100-200 mL) and transfer to FACS tube.
  4. FACS analysis



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.



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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