Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors

Authors

  • Sue Chow,

    1. Department of Pathology, Princess Margaret Hospital, Toronto, Ontario, Canada
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  • Harshna Patel,

    1. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
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  • David W. Hedley

    Corresponding author
    1. Department of Pathology, Princess Margaret Hospital, Toronto, Ontario, Canada
    2. Department of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada
    3. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada
    • Department of Medical Oncology and Hematology, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada
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Abstract

Cancer cells frequently show abnormal signaling via the mitogen activated protein kinase (MAP kinase) pathway due to increased activity of surface receptors for growth factors, or as a result of ras mutations. The development of potent anti-cancer agents that target this pathway prompts the need for analytical methods that allow pharmacodynamic monitoring of drug effects in patients during early phase clinical trial. We describe such a method, based on the activation of T-lymphocytes in undiluted peripheral blood using phorbol myristate acetate (PMA). Following rapid hypotonic lysis and formaldehyde fixation, activation of the MAP kinase pathway can then be demonstrated using phospho-specific antibodies that recognize the activated mediators MEK or ERK, followed by surface labeling with anti-CD3 to identify T-lymphocytes. This method was used to investigate the effects of a MEK inhibitor, U0126, and a new raf kinase inhibitor BAY 37-9751 in blood samples from normal donors. Dose-dependent inhibition of pERK activation was demonstrated for both agents. Furthermore, differential effects on pMEK activation allowed the molecular targets of the two inhibitors to be distinguished. In addition to monitoring drug effects in patients during treatment with inhibitors of the MAP kinase pathway, the general methodology described in this paper has the potential for wide application to the study of signal transduction at the single cell level using flow cytometry. Cytometry (Comm. Clin. Cytometry) 46:72–78, 2001. © 2001 Wiley-Liss, Inc.

The mitogen activated protein kinase (MAP kinase) signal transduction cascade is one of the major regulators of growth in animal cells (1, 2). The first component of this pathway consists of surface receptors for growth factors such as epidermal growth factor and GM-CSF. These receptors undergo tyrosine phosphorylation in their intracellular domains following binding with their respective ligands. Activated receptor tyrosine kinases can then activate ras proteins, which in turn activate divergent downstream signaling pathways including MAP kinase (2, 3). Individual elements of the MAP kinase pathway are raf, MEK, and ERK. Upon phosphorylation, each of these acquires protein kinase activity and is then able to phosphorylate and activate the next member of the signaling cascade. The final member, ERK (extracellular signaling regulated kinase) consists of two closely related proteins, ERK1 and ERK2, of 42kD and 44kD. Phosphorylated ERK1/2 (pERK1/2) translocates to the nucleus where it activates transcription factors involved in cell cycle regulation such as c-jun (2, 3).

Increased activity of the MAP kinase pathway due to aberrant receptor tyrosine kinase activation or ras mutations is extremely common in human cancers, and represents a major factor determining abnormal growth control (4, 5). Agents that inhibit this pathway show anti-tumor effects in experimental models, and have produced encouraging early results treating human cancers (6). This has become a very active area in the pharmaceutical industry, with many potent and specific new compounds now entering early phase clinical trial.

Most conventional cancer chemotherapeutic agents cause death in a dose-dependent manner by the production of non-specific DNA damage. These agents generally have a narrow therapeutic window, in that the effective anticancer dose is close to the maximum dose tolerated by normal tissues. In contrast, the optimum dose of a molecular targeted agent would be that which produces specific target inhibition. The biological effect of target inhibition will depend on its importance for the maintenance of tumor and normal tissue growth. For many agents, including compounds that inhibit signaling through the MAP kinase pathway, the effect might be cessation of growth, rather than cell death. Thus, although the introduction of molecular targeted agents offers the hope for more effective and less toxic cancer therapy, it requires a new approach to early phase clinical trial design.

Pharmacodynamic assays measure interactions between drugs and their molecular targets at the tissue level. Relative to the standard endpoints of tumor shrinkage and toxic side effects, they provide a more relevant measure of the effects of dose schedule using molecular targeted anticancer agents. Ideally, pharmacodynamic assays are applied to the malignant cell population. However, repeated sampling during the course of treatment is problematic except for diseases such as leukemia. Alternatively, drug effects on normal tissues can be measured as a surrogate marker. Peripheral blood lymphocytes can be readily brought into the cell division cycle, and thus have the potential to provide a surrogate marker for the effects of MAP kinase inhibitors in cancer patients. In this paper we describe the development of a sensitive assay based on the use of phospho-specific antibodies to activated ERK1/2 and flow cytometry.

MATERIALS AND METHODS

Peripheral Blood Samples

Peripheral blood samples using EDTA as the anticoagulant were obtained from normal donors, or as discards from the hematology laboratory according to institutional guidelines. For some experiments mononuclear cell suspensions were obtained by density gradient centrifugation on Ficol-Hypaque, and the concentration adjusted to 1×106/ml in αMEM medium containing 10% fetal calf serum (Cansera, Rexdale, ON).

Chemicals and Antibodies

Phorbol myristate acetate (PMA; #P8139) was obtained from Sigma, and used at a 40μM working dilution dissolved in 100% ethanol. The MEK inhibitor U0126 (7) was obtained from Cell Signaling Technology, Beverly MA (product #9903), and made up as a 10mM solution in methanol. The raf kinase inhibitor BAY 37-9751 was obtained from Bayer Pharmaceuticals (West Haven, CT) and made up as a 50mM solution in DMSO. BAY 37-9751 is a close structural analog of the raf kinase inhibitor BAY 43-9006, now entering phase I clinical trial. Phospho-specific rabbit polyclonal antibodies to ERK1/2 (#9101) and MEK (#9121) were obtained from Cell Signaling Technology, Beverly MA. A FITC-conjugated goat F(ab′)2 anti-rabbit IgG secondary antibody (#L43001) was obtained from Caltag (Burlingame, CA).

Lymphocyte Activation

PMA was added to isolated lymphocytes in tissue culture medium or to undiluted whole blood using a range of concentrations and stimulation times. For some experiments T-cells were activated by incubation with anti-CD3, followed by the addition of goat anti-mouse antibody to cross link the T-cell receptor, as previously described (8).

Western Blots

Protein extracts were prepared from isolated lymphocytes using lysis buffer (1% Triton X-100, 0.1% SDS, 50mM Tris (pH 8.0) 150 mM NaCl, 1mM phenylmethylsulfonyl fluoride, 0.1mM NaVO4, 0.1mM benzamidine, 5μg/ml leupeptin, and 5μg/ml aprotinin). Samples containing 25μg of total protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using the Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad, Mississauga, ON). ERK1/2 activity was detected using the phospho-specific rabbit polyclonal antibody obtained from Cell Signaling Technology. Secondary antibody containing the horseradish peroxidase detection system for chemoluminescence was used as recommended by the manufacturer (New England Biolab).

Immunofluorescence Labeling for pERK1/2 and pMEK

Fixation and permeabilization.

Cell suspensions were fixed using formaldehyde (10% Ultrapure EM Grade; Polysciences, Warrington PA), using a range of concentrations from 0.5% to 4%. Following fixation samples were resuspended in either methanol or ethanol at concentrations ranging from 70-100%. The final conditions that worked optimally are given in the Results section. Finally, samples were washed and resuspended in PBS containing 4% fetal bovine serum (FBS) at a final concentration of 1×106 cells in 100μl.

Antibody labeling.

Cell suspensions were labeled with the primary phospho-specific antibody at a range of concentrations for 15 min at room temperature, then washed in PBS containing 4% FBS and labeled using the FITC-conjugated goat F(ab′)2 anti-rabbit IgG secondary antibody. After washing, samples were labeled using an anti-CD3-PE antibody (IM 1282) obtained from Beckman-Coulter (Miami, FL).

Flow Cytometry

Flow cytometry was done using an Epic Elite cell sorter (Beckman-Coulter, Miami, FL). An air cooled argon laser operating at 20mW was used for excitation. Fluorescence signals were collected using bandpass filters centered at 525 and 575nm, with fluorescence compensation set to suppress FITC spillover into the PE channel.

RESULTS

Effects of PMA on pERK1/2 Determined by Western Blot

Using the phospho-specific antibody to pERK1/2, protein extracts from PMA-stimulated mononuclear cells produced a pair of strong bands on western blots, corresponding to the 42kD and 44kD forms of ERK. This effect was dose-dependent, with maximum stimulation using 40nM PMA. Unstimulated cells produced very faint 42/44kD bands, and no additional bands were observed indicating that the antibody specifically labels the phosphorylated forms of ERK1 and ERK2. Time course experiments illustrated in Figure 1A show that the activation of pERK1/2 by PMA occurred rapidly, and was sustained at a fairly constant level for 30 min. The raf kinase inhibitor BAY 37-9751 inhibited activation of pERK1/2 by 40nM PMA in a dose-dependent manner, with complete inhibition at a dose of 100μM BAY 37-9751 (Fig. 1B).

Figure 1.

A: Western blots showing the time course for ERK phosphorylation following activation with 40nM PMA. The two bands correspond to the 42kD and 44kD isoforms of ERK1/2. B: Western blots showing the effects of the raf kinase inhibitor BAY 37-9751 on ERK activation by PMA. Lanes show control, effects of 50μM BAY 37-9751 and 40nM PMA alone, and the effects of increasing concentrations of raf kinase inhibitor on PMA-induced ERK activation.

Measurement of pERK1/2 by Flow Cytometry

Fixation and permeabilization.

Protein phosphorylation is a dynamic process controlled by the enzymatic activities of kinases and phosphatases. In order to inhibit these processes rapidly, thereby preserving the phosphorylation states of ERK and MEK, fixation was done by adding formaldehyde to cell suspensions at 37°. A range of formaldehyde concentrations and fixation times was tested. Based on optimum fluorescence labeling and background labeling of samples stained with secondary antibody only, a concentration of 2% formaldehyde for 10 min was selected. This concentration was achieved by resuspending the cell pellet in 2% formaldehyde, or by adding the appropriate volume of the 10% solution to the cell suspensions held at 37°. Following fixation, the cells were pelleted by centrifugation and permeabilized by resuspension in methanol or ethanol. After testing a range of conditions, 1 ml 90% ice cold methanol for 30 min was determined to give the optimum results for antibody staining. Methanol was added while vortex mixing the tubes. As an alternative to resuspending the cell pellet, it was found that similar antibody labeling intensities were obtained when the samples containing formaldehyde were first cooled on ice for 2-3 min, followed by the addition of the appropriate volume of 100% methanol to give a final concentration of 90%.

Following fixation and permeabilization, the samples were either washed once with PBS containing 4% FBS and processed for antibody labeling, or stored at −20°. It was found that samples could be stored in 90% methanol for at least 4 weeks without significant loss of pERK1/2 immunofluorescence labeling or increase in non-specific background labeling relative to flow cytometry measurements made immediately after sample preparation.

Antibody labeling.

A preliminary dilution curve showed that saturation binding with the primary antibody was achieved at a dilution of 1/100, labeling for 15 min at room temperature. Using this concentration of primary antibody, the dilution curve for the secondary antibody indicated that 0.25μg (1μg = 1.25μl antibody) per 100μl sample gave optimum specific immunofluorescence labeling. Secondary antibody labeling was done at room temperature for 15 min. After labeling with the primary and secondary antibodies, the samples were washed once by resuspending in 2ml PBS containing 4% FBS. The effects of the number of washes for each antibody were investigated. It was found that no significant further reductions in non-specific background fluorescence were obtained using two or more washes after each antibody labeling.

Effects of PMA on pERK1/2

The effects of PMA on pERK1/2 activation in isolated lymphocytes were determined using flow cytometry. As illustrated in Figure 2, PMA activated pERK1/2 in a dose-dependent manner, with a plateau being reached at 40nM PMA. This concentration gave a relatively uniform level of pERK1/2 immunofluorescence labeling that was well separated from non-activated controls, and from activated samples where the primary antibody was omitted. Interestingly, Figure 2 shows that when low concentrations (e.g. 10nM) of PMA were used, individual cells appeared to be either maximally activated, or showed similar pERK1/2 values to those seen in the unstimulated control sample, resulting in a biphasic distribution pattern. Bivariate plots of pERK1/2 versus CD3 showed that activation by 40nM PMA was similar in T- and B-lymphocytes (Fig. 3).

Figure 2.

Effects of PMA and CD3 cross linking on activation of ERK1/2 measured by flow cytometry. The histograms bottom right are overlays of unstimulated control and 3 min following CD3 cross linking.

Figure 3.

Single parameter histograms (top) and dual parameter plots of phosphorylated ERK1/2 vs. CD3 (bottom). Lymphocyte gating on forward vs. orthogonal light scatter. Right panels show background staining of secondary antibody. Note that the extent of ERK activation by PMA is similar for CD3 positive and negative cells.

Effects of CD3 on pERK1/2.

Phorbol esters activate raf kinase via protein kinase C, rather than through cell surface receptor binding (9). Cross linking the T-cell receptor using anti-CD3 provides a more physiological stimulus that has been extensively used to study calcium fluxes by flow cytometry (8). Increases in pERK1/2 levels were detected in lymphocytes activated by anti-CD3 cross linked with a goat anti-mouse secondary antibody. However, the signal intensity was much less than that achieved with PMA; approximately 2-fold that of unstimulated controls, compared to approximately 30-fold using PMA (Fig. 2). Furthermore, CD3 stimulation of ERK1/2 was found to be transient, peaking at 2–3 min.

PMA Activates ERK via MEK

Due to the low and transient signal intensities produced, CD3 cross linking was considered to be insufficiently robust for use in a routine assay for activation of the MAP kinase pathway. To address concerns that the activation ERK by PMA might be occurring in part via other signaling pathways we examined the effects on MEK; the signaling element immediately upstream of ERK in the MAP kinase pathway. Two approaches were used. Firstly, the labeling method was modified by substituting a phospho-specific antibody to MEK, using the same general protocol described above. Secondly, U0126 and BAY 37-9751, which are specific inhibitors of MEK and raf kinase respectively, were tested for their effects on the activation of ERK by PMA.

After testing a range of labeling conditions, the final protocol used to measure phosphorylated MEK was identical to that for pERK1/2, with the antibody to pMEK being used at a 1/100 dilution. Activation of lymphocytes with PMA produced a large increase in pMEK (Fig. 4A). Addition of the MEK inhibitor U0126 inhibited the activation of ERK by PMA in a dose dependent manner, with close to 100% inhibition being produced at a concentration of 10μM; similar to that previously reported to inhibit MEK. No inhibition of pMEK activation by PMA was obtained using U0126, consistent with this agent being a specific MEK inhibitor. However, the raf kinase inhibitor BAY 37-9751 blocked activation of both MEK and ERK by PMA (Fig. 4A). The results from these experiments are therefore consistent with PMA activating ERK via raf/MEK, as illustrated in Figure 4B, without significant activation occurring through additional signal transduction pathways.

Figure 4.

A: Comparison of MEK (top) and ERK (bottom) activation by PMA, and effects of the inhibitors U0126 and BAY 37-9751. Note that PMA activates both MEK and ERK. This is inhibited by BAY 37-9751, whereas U0126 inhibits ERK but not MEK activation. These data are consistent with the linear pathway for ERK activation by PMA shown in B.

Development of Whole Blood Activation Assay

To test for inhibition of the MAP kinase pathway in patients receiving treatment with signal transduction inhibitors, it will be important to examine the response to PMA stimulation in undiluted whole blood, due to the potential loss of target inhibition during lymphocyte isolation. The development of a whole blood assay for MAP kinase activation presented a challenge because of the need to preserve the phosphorylation status of ERK (or MEK) while eliminating red blood cells. For example, fixation in 2% formaldehyde, which stabilizes pERK1/2 levels, rendered the red cells resistant to lysis. With lower formaldehyde concentrations the red cells could be lysed using ammonium chloride, but pERK1/2 labeling was then significantly compromised. None of the commercially available fixation and permeabilization reagents tested gave satisfactory results. However, it was found that hypotonic lysis of red cells following PMA stimulation yielded consistent, acceptable results.

One hundred microliters of whole blood with EDTA as the anticoagulant were dispensed to the bottom of two 5ml polypropylene Falcon tubes. The tubes were then placed in a 37° dry bath, and PMA added to one tube at a range of concentrations. After various time intervals, 2ml distilled water were added to each tube for 30 seconds, following which 220μl of 10x PBS were added. The tubes were immediately centrifuged at 1800g for 3 min, and the supernatant aspirated. The cell pellet was loosened by brief vortex mixing, and 100μl 2% formaldehyde added. The cells were fixed at 37° for 10 min, as for the isolated lymphocyte method, following which they were cooled on ice for 2-3 min. Nine hundred ml ice cold methanol were then added while vortex mixing, to give a final concentration of 90%. The tubes were then either stored at −20° or processed for pERK1/2 labeling.

Representative data using the whole blood activation assay are shown in Figure 5. A concentration of 400nM PMA was required to achieve maximum ERK activation, probably due to excess PMA binding in whole blood. However, using this concentration of PMA the fluorescence labeling intensities for pERK1/2 were comparable to those observed in isolated lymphocytes. Pre-incubation with U0126 and BAY 37-9751suppressed PMA-induced activation of pERK1/2 in a dose-dependent manner, with higher doses of each agent needed; presumably due to increased drug binding to lipids or proteins in whole blood.

Figure 5.

Whole blood activation assay to show effects of MEK inhibitor U0126. Dual parameter plots of pERK1/2 vs. CD3, using light scatter gating to include lymphocytes. Sample was pre-incubated with 100μM U0126 for 15 min, then activated with 400nM PMA for 10 min prior to red cell lysis.

Because samples for pharmacodynamic monitoring might be collected at inconvenient times, we examined the effects of prolonged storage of blood samples. Specific and non-specific antibody labeling intensities for PMA-stimulated and control samples were similar when the samples were examined fresh, or following up to 48 h storage at room temperature.

During red cell lysis and centrifugation, cells are diluted and potentially the effects of PMA on pERK1/2 are diminished due to the action of serine/threonine phosphatases that dephosphorylate ERK. To test this, we examined the effects of adding the phosphatase inhibitor okadaic acid (10μM; Calbiochem) at the time of red cell lysis. This did not result in a stronger pERK1/2 signal, indicating that significant dephosphorylation is unlikely to occur between hypotonic lysis and formaldehyde fixation.

Laboratory protocols for sample preparation, including the whole blood activation assay, are given in the Appendix.

DISCUSSION

Whereas conventional chemotherapy agents show greater anticancer effects and normal tissue toxicity with increasing drug doses, the effects of a molecular targeted agent are expected to reach a plateau once the drug target is maximally inhibited. The consequences of this inhibition on the growth of the tumor and normal host tissues would obviously depend on the importance of the drug target to the maintenance of cell function, but potentially could be less dramatic than those seen with chemotherapy drugs; producing for example growth arrest rather than an immediate reduction in tumor volume. Drug doses greater than those required to inhibit the specific target would increase the cost of treatment, and could potentially produce unwanted effects due to cross-reactivity with other molecular targets. For these reasons there is an increasing need for pharmacodynamic assays that are able to monitor drug effects at the tissue level during early phase clinical trials.

The assay described in this paper was developed for monitoring the effects of a novel raf kinase inhibitor, BAY 43-9006, now entering phase I clinical trial at our own hospital and in other institutions worldwide. However, it is clearly applicable to other agents that inhibit signaling via the ERK pathway. Although the assay is currently being applied to normal T-lymphocytes rather than to tumor cells, and therefore provides a surrogate marker for drug effect, we are in the process of adapting it for application to acute myeloid leukemia patient samples, which frequently show aberrant activation of the MAP kinase pathway (10). In this setting the assay would be used for direct monitoring of the malignant cell population during treatment with BAY 43-9006 or other agents.

A wide range of phospho-specific antibodies is now available from commercial sources. Although there is an extensive literature describing their use based on western blotting, so far as we can determine this is the first report studying the MAP kinase pathway by flow cytometry. Labeling methods for both pMEK and pERK1/2 were relatively easy to develop, and give robust signals that are well separated from those of unstimulated control cells. The availability of specific inhibitors such as U0126 and BAY 37-9751 provides additional quality control for the specificity of antibody labeling. Furthermore, since the flow cytometry assay correctly identified the molecular targets for these compounds, as illustrated in Figure 4, it has potential for screening new compounds for inhibitory effects on defined elements of the MAP kinase pathway within the cellular and/or tissue context.

Relative to established techniques such as western blotting, single cell measurements of phosphorylated proteins by flow cytometry has potential for a wide range of new research applications. For example, the biphasic response to low concentrations of PMA observed in Figure 2 suggests that the activation of pERK1/2 involves an all-or-none biological response, similar to that recently reported by Ferrell and Machleder to occur in Xenopus oocytes treated with progresterone (11). This question is of fundamental importance to understanding the biochemical basis of cell signaling in intact cells. Whereas the single cell immunoblot measurements described by these authors would be difficult to perform routinely on mammalian cells, this type of experiment could be readily done using the flow cytometry technique. In addition to examining the effects of PMA on T-cell activation, we have begun to adapt the flow cytometry method for studying leukemia patient samples and normal human bone marrow. Preliminary results confirm that the activation of CD34 positive myeloid progenitor cells using cytokines such as kit-ligand (stem cell factor) can be readily detected by increases in pERK1/2, the magnitude of this effect being comparable to that seen in PMA activated T-cells. Furthermore, we anticipate that the flow cytometry technique can be further developed to study other signal transduction pathways, such as the phosphatidylinositol 3-kinase/PKB/Akt anti-apoptosis pathway (12–13). Flow cytometry assays that are sensitive to the activation of multiple signaling pathways, and capable of studying heterogeneous cell populations defined by immunophenotypic markers, would offer a powerful new approach to basic research in signal transduction, as well as finding novel clinical applications.

Acknowledgements

We wish to thank James W. Jacobberger, Case Western Reserve University, for much practical advice about intracellular antigen staining. Gideon Bollag and John Lyons of Onyx Corporation, and Steve Wilhelm of Bayer Pharmaceuticals made many helpful suggestions during the development of this technique. The authors also wish to thank Bayer Pharmaceuticals for supplying the raf kinase inhibitor BAY 37-9751, and for financial help with this work.

APPENDIX

Activation of ERK and MEK in Isolated Lymphocytes

  • 1.Resuspend cells at 1×106/ml in tissue culture medium such as αMEM plus 10% fetal bovine serum (FBS), 37° water bath or dry bath.
  • 2.Activate for 10 min using 40nM phorbol myristate acetate (Sigma #P8139; 40μM working dilution in ethanol). Non-activated control cells.
  • 3.While maintaining sample at 37°, fix by adding 10% formaldehyde (Ultrapure EM Grade; Polysciences) to give a final concentration of 2% formaldehyde. Fix at 37° for 10 min.
  • 4.Place tubes on ice for 2–3 min, then add 100% ice cold methanol with gentle vortex mixing, to give a final concentration of 90% methanol. Hold on ice for 30 min, then process for antibody labeling or store at −20° for up to 4 weeks.
  • 5.Centrifuge tubes, aspirate, and wash once using 2ml PBS containing 4% FBS. Centrifuge and resuspend at 106 cells in 100μl PBS plus 4% FBS.
  • 6.Label with phospho-specific polyclonal antibody to ERK1/2 (Cell Signaling Technology #9101) or MEK (Cell Signaling Technology #9121). Both primary antibodies used at a 1/100 dilution. Label for 15 min at room temperature, then wash once using 2ml PBS plus 4% FBS.
  • 7.Resuspend at 106 cells in 100μl PBS plus 4% FBS. Add 0.25μg (1μg = 1.25μl) FITC-conjugated goat F(ab′)2 anti-rabbit IgG secondary antibody (L43001; Caltag). Label at room temperature for 15 min. If desired, PE-conjugated anti-CD3 can be added to the sample at the same time as the secondary antibody, in order to identify T-lymphocytes. Wash once in 2ml PBS plus 4% FBS.
  • 8.Run on flow cytometer.

Troubleshooting

Effects of PMA similar in CD3 positive and negative cells. Since the primary antibody is an intact rabbit polyclonal, it can bind to surface Fc receptors. Expect to see 20-30 fold increase in pERK1/2 and 10-20 fold increase in pMEK with PMA activation. Use additional controls omitting primary antibodies if needed. The specific MEK inhibitor U0126 (Cell Signal Technology #9903), added at a final concentration of 10μM for 15 min at 37° prior to PMA treatment, blocks the activation of pERK1/2 but not pMEK.

Whole Blood Activation Assay

  • 1.Use EDTA as the anticoagulant. Potentially whole blood samples can be held overnight or longer at room temperature. Dispense 100μl undiluted blood to the bottoms of two 5ml polypropylene Falcon tubes (or similar). Avoid contaminating the sides of the tubes with blood. Place in 37° water bath or dry bath.
  • 2.Add a final concentration of 400nM PMA to one tube (NB - tenfold higher concentration than for isolated lymphocytes). Keep at 37° for 10 min.
  • 3.Add 2ml distilled water to each tube. After 30 seconds (time critical) add 220μl of ×10 strength PBS to each tube and vortex immediately.
  • 4.Immediately centrifuge at 1800g for 3 min. Aspirate the hemoglobin-containing supernatant as thoroughly as possible.
  • 5.Vortex to loosen up the cell pellet, then add 100μl 2% formaldehyde to the bottom of each tube and mix. Hold at 37° for 10 min.
  • 6.Place on ice for 2–3 min, then add 1ml ice cold 100% methanol with vortex mixing. Hold on ice for 30 min, then process for antibody labeling as above or store at −20° for up to 4 weeks.

Ancillary