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The fundemental utility of flow cytometry is the ability to discriminate cell types based on the differential expression of surface proteins. The addition of phospho-specific antibodies now allows the analysis of signaling responses within populations resolved by surface markers. Here we show sample data where mixtures of cells are stimulated and the signaling responses of multiple cell types are followed simultaneously. All of the data shown here was collected on a four color flow cytometer (FACSCalibur, Becton Dickinson).
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Signaling responses of murine splenocytes to a panel of stimuli. The spleen of a BALB/c mouse was homogenized in cold PBS + 5mM EDTA, spun down, and cultured in RPMI + 10% FCS + PSQ at 37C for 1.5 hours before stimulation. Stimulation time was 15 minutes with recombinant murine cytokines (Peprotech) or lipopolysaccharide (E. coli 0127:B8) followed by formaldehyde fixation and permeabilization with methanol. Samples were split to allow staining with different antibody combinations (AB from Becton Dickinson). Surface gating was as shown. Each histogram shows the staining of the unstimulated sample as a grey histogram and the stimulated sample as a red line histogram. (A.) B cell pStat1 (Y701) response to interferon gamma. (B.) T cell pStat3 (Y705) response to IL-6. (C.) Monocytes/DC pStat5 (Y694) response to GM-CSF. (D.) Monocytes/DC pp38 (T180/Y182) response to LPS. |
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| Figure courtesy of Matt Hale and Peter Krutzik, Nolan Lab, 2004 | |
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| pStat3 (Y705) response of murine splenic dendritic cells to IL-10. Splenocytes from a BALB/c mouse were cultured in RPMI + 10% FCS + PSQ for 1.5 hours prior to 15 minute stimulation. The cells were formaldehyde fixed and methanol permeabilized. Intact cells were gated on scatter properties and surface gating was as shown. The grey histogram is the unstimulated culture and the red line histogram is the culture stimulated with murine recombinant IL-10 (100ng/ml). |
| Figure courtesy of Matt Hale, Nolan Lab, 2004 |
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| pStat6 responses of human natural killer cells
to a panel of stimuli. 1 x 10^6 PBMCs (nondepleted)
were treated with the indicated cytokine (200 ng/ml) for 15 minutes
at 37C formaldehyde fixed and methanol permeabilized. The cells
were then simultaneously stained with CD56-PE (clone B159), CD11b-cychrome
(clone ICRF44), and phospho-STAT6 (Y641)-AX647 (clone 18). Surface
gating was as shown. Figure courtesy of Omar Perez, Nolan Lab 2004. |
