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Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Although these studies were able to discriminate cell types based on surface marker expression, separation between positive and negative populations often decreased, especially when methanol permeabilization was used (5). As we have begun to apply phosphospecific flow cytometry to more immunological cell types, we have encountered difficulties while trying to stain for surface Ags after cells have been fixed and/or permeabilized. In particular, staining of B cell progenitors in the bone marrow has proven difficult due to poor CD43 and CD24 staining, both of which are critical to separation of early B cell populations. Identification of mast cells from splenic and peritoneal populations has also been hindered by poor Fc
R1a and c-Kit resolution. Thus, much effort has been put into finding and characterizing Abs that work in the context of phosphospecific flow cytometry. We believe that as researchers increasingly apply the technique to rare cell types and novel organ systems, cell subset identification will be of critical importance. Therefore, we decided to undertake the current study to generate a comprehensive profile of the effects of fixation and permeabilization on surface marker Ab-staining characteristics to provide both a resource of data and a set of guidelines to follow when applying the technique.
Due to the transient nature of phosphorylation events, cells must be fixed rapidly after stimulation to freeze the phosphorylation status of proteins. Also, to analyze phosphorylation at the single-cell level, cells must be permeabilized to allow phosphospecific Abs access to relevant phosphoepitopes inside the cell (1). Fixation and permeabilization are thus absolutely required for the technique in its current state of application (9, 10, 11). Although many techniques have been used to analyze phosphoproteins by flow cytometry (2, 3, 4, 5, 6, 8, 12, 13, 14, 15, 16, 17, 18), we recently showed that formaldehyde fixation followed by methanol permeabilization (F/M)3 was superior for the analysis of many key signaling molecules of the immune system, especially those of the STAT protein family (7). In addition to providing high sensitivity and signal-to-noise ratios, methanol has an advantage over saponin permeabilization in that phosphoepitopes are stable at –80°C for prolonged periods (months) when stored in methanol (7). This is especially important for experiments involving clinical samples, where samples can be collected over weeks or months, then stained and analyzed simultaneously.
Because multiple steps are required to prepare cells for analysis by phosphospecific flow cytometry, the simplest procedure would be to stain cells for surface markers and intracellular phosphoproteins simultaneously, after fixation and permeabilization. Therefore, in the context of intracellular detection of phosphoproteins, we set out to characterize the effects of formaldehyde fixation and methanol or saponin permeabilization on the detection of cell surface Ags and the maintenance of light scatter properties. Most Abs worked well after F/M treatment, although the separation between positive and negative populations decreased in nearly all cases. In addition, optimal titration conditions differed significantly after F/M treatment. For those Abs negatively affected by permeabilization, we tested whether a stepwise staining of surface markers after fixation, but before permeabilization, could rescue the staining efficiency for these Ags. After screening >35 Abs, we applied surface staining in combination with intracellular phosphoprotein analysis to characterize the immune cell subsets that were responsive to IL-6 and IL-10 treatment and to analyze CD4+ and CD8+ T cell differences in cytokine response. We show in this study the many parameters that must be taken into consideration when applying the technique to primary cell populations and provide a basic framework for researches interested in applying phosphospecific flow cytometry to their complex tissues of interest, particularly those of the immune system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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All surface and phosphospecific Abs were provided by BD Pharmingen and are summarized in Table I. Abs were supplied conjugated to FITC, Alexa Fluor 488 (Ax488), PE, Cy5PE, PerCP, Cy5.5 PerCP, allophycocyanin, or Ax647. The Ab concentration was 0.2 mg/ml, except for FITC conjugates, which were at 0.5 mg/ml. Phosphospecific Abs against Stat3 (Y705, clone 4) and Stat5 (Y694, clone 47) were conjugated to Ax647 and were used at optimal titrations.
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Surface marker Ab analysis
Male BALB/c and C57BL/6 mice were obtained from the Stanford Animal Core Facility in-house breeding colony and were used at 6–12 wk of age. Animals were treated in accordance with university and Administrative Panels on Laboratory Animal Care guidelines. After death, peritoneal cavity cells were obtained by flushing the peritoneum with MEM containing 5% FBS. Spleens were removed and homogenized in MEM and 5% FBS; bone marrow cells were obtained by flushing tibia and femurs with the same buffer. All cells were then passed through a 70-µm pore size mesh, pelleted, and resuspended at room temperature in RPMI 1640 containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine at 5 x 106 cells/ml. Cells were then cultured at 37°C for 2 h before surface marker analysis or stimulation with cytokines for combined surface and intracellular phosphoprotein analysis.
For surface marker analysis alone, staining with Abs was performed at multiple points. For unfixed samples, cells were pelleted out of the culture medium and resuspended in staining medium (PBS with 0.5% BSA and 0.02% sodium azide) at 107 cells/ml. Staining was performed in 100-µl volumes (106 cells). Abs were added at various dilutions, and cells were stained for 25–30 min. The cells were washed with 15 vol of staining medium, pelleted, and resuspended for flow cytometric analysis. For formaldehyde fixation, formaldehyde was added directly to culture medium to a final concentration of 1.6% and incubated for 10 min at room temperature. Cells were pelleted, and then resuspended in staining medium and stained as described above. For samples that were permeabilized with methanol after this staining step, cells were washed with PBS instead of staining medium and resuspended directly in ice-cold 100% methanol after pelleting. Cells were incubated at 4°C for 10–30 min before washing twice with staining medium and analyzing by flow cytometry. Finally, for cells that were both fixed and permeabilized before staining, the formaldehyde-fixed cells were pelleted and resuspended directly in 100% methanol. They were incubated for 10–30 min at 4°C before being washed twice with staining medium and stained with Abs as described above. When cells were permeabilized with saponin, 0.1 or 0.5% solutions of saponin in staining medium were substituted for methanol. Also, saponin was present during staining to maintain cell permeability, because saponin permeabilization is reversible.
Simultaneous phosphospecific and surface Ab staining
Phosphospecific flow cytometry was performed as previously described (7). Briefly, splenocytes in RPMI 1640 with 10% FBS were stimulated with the indicated cytokines for 15 min at 37°C before fixation with 1.6% formaldehyde for 10 min. The cells were then pelleted, resuspended in ice-cold methanol, and incubated for 15–30 min at 4°C. The cells were washed twice with staining medium, resuspended at 107 cells/ml, and stained with a mixture of Abs including CD11b, TCR
, B220 and phospho-Stat (pStat3)(Y705), or TCR, CD4, CD8, and pStat5(Y694). Cells were stained for 30 min, then washed with staining medium and resuspended before FACS analysis.
Flow cytometry
All flow cytometry was performed on a FACSCalibur instrument equipped with a 633-nm helium-neon laser for allophycocyanin and Ax647 excitation. After acquisition, data were analyzed with a FlowJo (TreeStar). Three-dimensional plots were generated by exporting the scale values of 2000 cells to Spotfire DecisionSite8.0. Median fluorescence intensity (MFI) values were determined for both positive and negative populations of cells. The efficacy of Abs was assessed by determining the ratio between the MFI of the positive population and the MFI of the negative population. Larger ratios indicate better separation of populations. The percent loss upon fixation and permeabilization was calculated as % loss = 100 – 100 x (MFIpos/MFIneg)fixed/(MFIpos/MFIneg)unfixed. For phosphoprotein analysis, the fold change upon stimulation was calculated as the MFIstimulated/MFIunstimulated ratio for the phosphospecific Ab fluorescence channel (FL4).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To highlight the utility of phosphospecific flow cytometry in analyzing immune cell signaling, we applied the technique to analyze IL-2, IL-6, IL-10, and IL-15 signaling in murine splenocytes. First, we treated splenocytes with IL-6 and IL-10 ex vivo and performed four-color flow cytometry with CD11b, TCR, B220, and pStat3(Y705) Abs (Fig. 1). After careful screening, we have found these surface Abs to maintain staining characteristics after F/M treatment (see Table I).
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, and CD11b to highlight the importance of the multidimensional aspect of flow cytometry (Fig. 1A). The B220 vs pStat3 plot reveals that B cells have a heterogeneous response to IL-6, but respond relatively uniformly, and strongly, to IL-10 with pStat3 induction. However, for IL-6, it is clear that B220– cells also respond, and do so with a larger increase in pStat3 levels. Plotting TCR vs pStat3 shows that T cells make up the majority of these B220– cells that are responsive to IL-6. The CD11bint population appears to contain two distinct subsets, one that is responsive to both IL-6 and IL-10 (the intermediate high cells, just above the quadrant line), and one that responds only to IL-10 (intermediate low cells, below the quadrant line). These intricacies are not accessible to standard biochemical techniques and require the analysis of samples at the single-cell level.
In addition, we sought to characterize the difference between CD4+ and CD8+ T cells in response to IL-2 and IL-15. Thus, we stained cells with TCR, CD8, CD4, and pStat5(Y694) (Fig. 1B). The plot of CD4, CD8, and pStat5 shows that a fraction of CD4+ cells respond to IL-2 and IL-15, whereas CD8+ cells uniformly respond to IL-15, but not to IL-2. Some CD4–CD8–TCR– cells also respond to IL-15 (most likely macrophages and dendritic cells). By resolving cell types on a single-cell basis, one begins to appreciate the underlying heterogeneity that is often present in seemingly uniform cell types. However, these forms of analysis require careful optimization of surface markers, such that cell types analyzed with phosphoprotein staining techniques correspond to those analyzed by standard staining and biochemical techniques.
Effects of fixation and permeabilization on surface marker Ab binding
To evaluate the effects of formaldehyde fixation and methanol permeabilization on surface marker analysis, we compared the staining characteristics of the tested Abs on live, intact cells and F/M-treated cells. Two parameters were used to assess Ab efficacy: the separation between positive and negative populations (e.g., the ratio of the MFIs of the two populations) and the percentage of cells that are positive. To obtain optimal separation, the surface Ab concentration during staining must be titrated. Thus, we titrated 36 different Abs against multiple classes of Ags on murine splenocytes, from functional receptors such as TCR and MHC to less well-characterized Ags such as CD24 and Ly6C, on both live cells and F/M-treated cells. The results of this extensive profile are summarized in Table I. It should be noted that although we use the term surface marker analysis for both live cells and fixed/permeabilized cells, addition of Abs against surface Ags to permeabilized cells will also measure intracellular stores of the Ag. We have found that most typical surface markers are present at very low concentrations inside cells, although some markers, such as IgG and CD69, may be detected before they are actually expressed on the cell surface. One must always be aware of this caveat when staining surface markers after permeabilization.
Abs were scored on a functional basis for the ability to resolve positive and negative populations as follows: negative (–) indicates no separation and cells cannot be discriminated based on this marker alone; single positive (+) indicates a <0.5 log difference, such that positive cells can be discriminated, but not clearly from this marker alone, and gating requires extra markers for accurate analysis; double positive (++) indicates a 0.5–1.5 log difference, such that positive cells can be discriminated based on this surface marker alone as long as a significant portion of the total population is positive, e.g., >5%; and triple positive (+++) indicates a >1.5 log difference, so that positive cells can be gated clearly on this marker alone even when they are present at very low numbers, e.g., <1% of the total cells. We used this scoring scheme to emphasize which Abs were effective after F/M (++ or +++) vs those that were difficult to use or were completely unusable (+ or –). Many Abs that scored +++ for both unfixed and F/M-treated cells actually showed a large decrease in separation after F/M (see B220 below). However, this loss does not affect simple identification of positive cells and therefore does not decrease the utility of the Ab for subsetting complex populations. Gr-1, Ly6C, and CD24 were difficult to score because of their broad positive populations. In some cases, only portions of positive subpopulations were lost, whereas other subpopulations were maintained. Particular attention should be paid to such Abs. The differential results displayed in Table I suggest careful analysis of these Abs and their titration levels before application to phosphoprotein characterization.
To highlight the differences between effective and ineffective Abs, we examined three Abs of each type more closely: B220, TCR, and MHC I-Ad (Fig. 2A) and CD24, Ly6C, and MHC I-Ab (Fig. 2B), respectively. Among the more effective Abs (Fig. 2A), TCR recognition is largely unchanged after F/M, with the optimal titration remaining at 1 µl, the percentage of positive cells at 35%, and only 50% loss in the positive to negative ratio. B220 recognition is also maintained, but 20-fold less Ab is required to obtain optimal separation between positive and negative populations after fixation and permeabilization. In this study, >80% of the separation is lost after fixation, yet positive cells are still 30-fold brighter than negative cells. Most of the reduction in separation is caused by a large increase in the MFI of negative cells, because the positive population has nearly identical MFI values on both fixed and unfixed cells at any given Ab titer. We attempted to decrease background binding levels by using BSA, FBS, and Ab isotypes as blocking reagents, but none of these reagents significantly enhances surface marker analysis (data not shown). Therefore, it appears that even markers that remain effective are negatively affected by F/M treatment and require careful titration for optimal separation.
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Intracellular Ags as source of background staining
It appeared that F/M treatment tended to increase background staining levels of nearly all Abs tested. This could be due to specific effects of methanol on intracellular and surface epitopes or to general surface Ab nonspecificity when introduced to the interior of cells. Therefore, we tested the effects of saponin permeabilization on surface marker Ab binding characteristics (Fig. 3). Although saponin permeabilization is not as effective for phosphoprotein analysis, we sought to determine whether cell permeabilization alone was sufficient to increase surface Ab background binding levels (19). Similar to methanol permeabilization, TCR, B220, and MHC I-Ad staining was maintained after saponin permeabilization, but showed increased background, decreased positive signals, decreased positive to negative ratios, and generally a reduction in optimal titrations.
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Rescue of poorly resolved surface markers by sequential staining techniques
Abs were first tested by staining after F/M permeabilization, because that is the simplest technique, requiring that cells be stained only once for both surface and intracellular Ags. In most cases, it appears this technique works with appropriate titration, but there are clearly instances (CD24, Ly6C, and MHC I-Ab) where the staining is ineffective after F/M. Thus, we tested whether sequential staining techniques could rescue recognition of these Ags (Fig. 4). As is often used for cytokine analysis, we first fixed cells with formaldehyde, stained them with appropriate titrations of surface Abs conjugated to FITC, then permeabilized them with methanol (Fig. 4, third column). As a comparison, cells were left unfixed (first column), fixed, and stained without subsequent permeabilization (second column) or were fixed, permeabilized, and then stained as described in Fig. 2 (fourth column).
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Surprisingly, MHC I-Ab staining could not be restored by sequential staining techniques (Fig. 4). In this study, formaldehyde fixation alone was sufficient to hamper detection and separation of positive from negative cells. Permeabilization with either saponin or methanol did not affect the separation.
Thus, it appears that both formaldehyde fixation and methanol permeabilization can act separately or together in negatively affecting the discrimination of immune cell types via surface markers. However, sequential staining of cells before permeabilization but after fixation can rescue the binding characteristics of many of these Abs and provides a method for analysis of "difficult" surface Ags. In addition, this technique avoids staining of intracellular Ag stores and may therefore provide results closer to those obtained with live, intact cells.
Fluorophore choices for sequential staining
Because sequential staining techniques were able to rescue two of three Ags that could not be analyzed after methanol treatment, the technique may become more widely used as phosphospecific flow cytometry techniques are applied to novel cell types. However, we were concerned that due to the dehydrating and denaturing effects of methanol, some fluorescent proteins, such as PE, PerCP, and allophycocyanin, might be destroyed if added before methanol. Therefore, we performed sequential staining of murine splenocytes with a B220-specific Ab conjugated to the small molecules Ax488, FITC, and Ax647; to the protein fluorophores PE, PerCP, and allophycocyanin; and to a tandem of small molecules on a protein fluorophore Cy5.5 PerCP (Fig. 5).
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Ab clone selection
We next tested whether particular Ags, such as CD4 or CD25, are altered by F/M treatment in such a way that all Abs directed against them are equally affected, or whether different Ab clones might be superior after fixation and permeabilization (Fig. 6). Four different FITC-conjugated Ab clones against CD4, RM4-5, GK1.5, H129.19, and RM4-4, were compared before and after F/M treatment. Clone RM4-5 was superior to all other clones, with only a 35% loss in signal, whereas RM4-4 was worst, with about an 80% loss (Fig. 6B). GK1.5, H129.19, and RM4-5 are competitive for their epitopes when added simultaneously, whereas RM4-4 binding is not affected by these other mAbs (according to the manufacturer’s data sheets). It therefore appears that the location of the RM4-4 epitope on the CD4 molecule may be modulated to a greater extent than the epitope recognized by the other clones.
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-chain) were tested (Fig. 6C). Clone PC61 was tested conjugated to both PE and Cy5.5PerCP, whereas clone 7D4 was conjugated to FITC, and clone 3C7 to PE. As was the case for CD4, the clones against CD25 responded differently to F/M treatment. PC61 was the least affected, although the Cy5.5PerCP-conjugated Ab was slightly worse than the PE-conjugated version, and 3C7 was particularly poor. Therefore, different clones against the same Ag should be tested when initial attempts at surface marker analysis fail. Testing other clones may yield better separations, especially if the clones are directed against different epitopes on the Ag.
Scatter properties of cells in immune organs after F/M treatment
Although most discrimination of immunological cell types is accomplished via surface marker analysis, many gating strategies use initial gates based on cell size and granularity, measured by forward scatter (FSC) and side scatter (SSC) properties, respectively. We therefore examined how F/M treatment affected scatter properties in multiple immunological organs, including the spleen, peritoneal cavity, and bone marrow (Fig. 7). B cells (B220+), T cells (TCR+), monocytes/macrophages/neutrophils (CD11b+ cells), and dendritic cells (CD11c+) were gated according to surface markers. These populations were then analyzed for FSC and SSC characteristics both before and after F/M treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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60% of B cells. By typical biochemical techniques, the response to IL-6 would appear about half that to IL-10. Yet, such an assessment would be incomplete and inaccurate, because it is clear that 60% of the cells show a maximal response, equivalent to the response to IL-10. Such detailed analysis will help reveal differences in subsets never before identified and will lead to a better understanding of the specific role of each cell type in the immune system signaling network. To this end, we have recently used these techniques to characterize the subset-specific differences in murine immune cell signaling both in vitro and in vivo and found striking comparisons between the two. Thus, phosphospecific flow cytometry is a powerful tool for analyzing immune cell signaling events, but has three major requirements for successful application (1). First, transient signaling events must be rapidly terminated or frozen at the time interval of choice (because many of the events decay on the time scale of minutes). Second, cells must be permeabilized efficiently for analysis with phosphospecific Abs. Third, to apply the technique to heterogeneous cell populations, surface Ags must also be stained to identify cell subsets of interest. Although several studies have used surface stains in parallel with phosphoprotein detection, they have primarily used saponin permeabilization and stained the surface Ags before saponin treatment (2, 3, 17). As mentioned previously, saponin permeabilization is limiting to the technique, because samples must be processed immediately after treatment, thereby making studies that require sample collection over several days less consistent. In addition, saponin is inferior to methanol permeabilization for several key phosphoproteins of immunological signaling pathways, particularly the STAT protein family (7).
However, our studies and others that have examined cytokine signaling using methanol permeabilization have found that it was more difficult to identify cell types based on surface marker characterization (4, 5, 20). It appeared that separation between positive and negative populations was greatly reduced when cells were stained after permeabilization, and fluorophore choices were limited when surface stains were applied before methanol treatment. Therefore, as with many techniques, one must assess which aspect of the experiment is more critical: the phosphoprotein analysis or the cell subset identification.
In this work we sought to answer whether surface markers can still be effectively analyzed after F/M treatment or if the majority of surface Abs are rendered unusable. In Table II, we summarize the critical features of Ab staining that were used to validate Abs for this platform. To our surprise, after screening >35 different murine surface marker Abs, we found that 80–85% identified the appropriate cell subsets with proper percentages and adequate separation. However, nearly all Abs tested suffered decreased separation between positive and negative populations, typically 80–90% (e.g., about 1 log on a standard FACS plot). In addition, the optimal titer usually decreased from that used on live cells, sometimes 5- to 10-fold (Fig. 2). We found that the decrease in resolution was primarily caused by an increase in background of negative cell populations, and that this increase was evident in all permeabilized cells regardless of the agent used (Fig. 3). However, it appears that the degree of permeabilization may affect Ab staining characteristics, with more harsh reagents such as methanol leading to larger increases in background. Because surface marker Abs are screened on live cells with intact membranes, it is impossible to predict a priori what the effect of exposing intracellular Ags will be. For several Abs that still worked after treatment with <0.5 log separation between populations (e.g., CD3, CD40, CD43, and CD49b), resolution was greatly enhanced if used in parallel with other markers to generate two-dimensional plots (e.g., CD3 vs B220).
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For the Abs with poor staining characteristics, we were able to restore adequate resolution of populations by staining for surface markers after formaldehyde treatment, but before methanol permeabilization (Fig. 4). However, this technique is limited, in that only small molecule fluorophores (Ax 488, Ax647, FITC, etc.) and possibly allophycocyanin can be used. PE and PerCP and their tandems were destroyed by methanol (Fig. 5) (4). Because the small molecule dyes are optimal for conjugation and sensitivity for phosphospecific Abs, using these dyes for surface markers hinders multidimensional staining, especially in rare cell types that require several markers to identify (7). Finally, staining before methanol limits later staining combinations, because one channel will effectively be removed, eliminating a key advantage of storage in methanol for later analysis with several Ab mixtures.
After screening >100 phosphospecific Abs for this platform, we believe that obtaining optimal phosphoprotein staining is most critical to successful application of the technique. Although several Ags were difficult to stain after F/M treatment, we believe that with screening of more Ab clones against multiple epitopes on the same Ag, Abs against all of the critical surface markers can be found within the available commercial Ab pool (see Fig. 6). It should be noted that our group has found human Ags to be more difficult to analyze after F/M treatment (data not shown). This may be due to differences between murine anti-human and rat anti-mouse Abs and the epitopes they tend to recognize.
In this study we provide a comprehensive profiling of most major surface molecules present on murine immune cells for analysis in conjunction with intracellular phosphoprotein staining. This serves as a starting point for researchers interested in applying phosphospecific flow cytometry to novel, complex populations of cells that require surface marker analysis. By understanding the parameters critical to successful surface staining and validation of new Abs, we expect that these results will simplify application of the technique as an alternative system for biochemical analysis of signaling networks in complex populations of immune lineage cells.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Heart, Lung, and Blood Institute Contract N01-HV-28183 and National Institutes of Health Grant AI35304. P.O.K. was supported by a Howard Hughes Medical Institute predoctoral fellowship. 
2 Address correspondence and reprint requests to Dr. Garry Nolan, Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, 269 Campus Drive, Center for Clinical Sciences Research 3205, Stanford, CA 94305. E-mail address: gnolan{at}stanford.edu
3 Abbreviations used in this paper: F/M, formaldehyde fixation and methanol permeabilization; Ax, Alexa Fluor; FSC, forward scatter; MFI, median fluorescence intensity; pStat, phospho-Stat; SSC, side scatter.
Received for publication March 10, 2005. Accepted for publication May 30, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation. J. Immunol. 172: 4235-4244. 
. Clin. Exp. Immunol. 126: 456-460. [Medline]
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 P. Trampont, L. Zhang, and K. S. Ravichandran ShcA Mediates the Dominant Pathway to Extracellular Signal-Regulated Kinase Activation during Early Thymic Development Mol. Cell. Biol., December 1, 2006; 26(23): 9035 - 9044. [Abstract] [Full Text] [PDF]
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S. L. Fanning, T. C. George, D. Feng, S. B. Feldman, N. J. Megjugorac, A. G. Izaguirre, and P. Fitzgerald-Bocarsly Receptor Cross-Linking on Human Plasmacytoid Dendritic Cells Leads to the Regulation of IFN-{alpha} Production J. Immunol., November 1, 2006; 177(9): 5829 - 5839. [Abstract] [Full Text] [PDF]
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L. S. Berenson, M. Gavrieli, J. D. Farrar, T. L. Murphy, and K. M. Murphy Distinct Characteristics of Murine STAT4 Activation in Response to IL-12 and IFN-{alpha} J. Immunol., October 15, 2006; 177(8): 5195 - 5203. [Abstract] [Full Text] [PDF]
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M. Long and A. J. Adler Cutting Edge: Paracrine, but Not Autocrine, IL-2 Signaling Is Sustained during Early Antiviral CD4 T Cell Response J. Immunol., October 1, 2006; 177(7): 4257 - 4261. [Abstract] [Full Text] [PDF]
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 G. P. Nolan Deeper Insights into Hematological Oncology Disorders via Single-Cell Phospho-Signaling Analysis Hematology, January 1, 2006; 2006(1): 123 - 127. [Abstract] [Full Text] [PDF]
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P. O. Krutzik, M. B. Hale, and G. P. Nolan Characterization of the Murine Immunological Signaling Network with Phosphospecific Flow Cytometry J. Immunol., August 15, 2005; 175(4): 2366 - 2373. [Abstract] [Full Text] [PDF]
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, unfixed cells; 
, F/M-treated cells). Numbers over the bar graphs indicate the percent loss upon F/M treatment. C, Anti-CD25 clones PC61, 7D4, and 3C7 were tested in BALB/c thymocytes. PC61 was used conjugated to PE and Cy5.5PerCP to compare the effects of the fluorophore on Ab accessibility and binding capacity. Note that the y-axis has a logarithmic scale for CD25. The mean and SD for each clone were calculated from at least three experiments.