Sample Data: 1 and 2 Color Flow Cytometric Analysis of
Phospho-epitopes with Correlation to Western Techniques
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| Comparison of flow cytometric analysis of phospho-epitopes
and western techniques. (A.) Cultures of the Jurkat human
T cell line were left unstimulated, or treated with PMA (50nM),
the combination of PMA and ionomycin (50nM PMA and 1uM IO), or
anisomycin (2ug/ml) for 10 minutes at 37C. Cells were then divided
and lysed for Western blot analysis or fixed with formaldehyde
and permeabilized with methanol for flow cytometric analysis
with pERK1/2 Alexa 488, pp38 Alexa 647, or pJNK Alexa647. Unconjugated
forms of these antibodies were used for Western blots. Unstimulated
samples appear as open traces in the FACS plots. (B.) Cultures
of the human monocytic cell line U937 were left unstimulated
or treated with recombinant human interferon gamma (50ng/ml),
IL-4 (10ng/ml), or GM-CSF (10ng/ml) for 10 minutes at 37C. The
cells were split as above, and analyzed with pStat1 Alexa488,
pStat5 Alexa488, and pStat6 Alexa647. |
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| Krutzik,
P.O. and G.P. Nolan, Intracellular phospho-protein staining techniques
for flow cytometry: monitoring single cell signaling events.
Cytometry, 2003. 55A(2): p. 61-70. |
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| Flow cytometric analysis of phospho-epitopes directly correlates
to western techniques. Human monocytic cell line U937 cells
were treated with increasing amounts of human recombinant interferon
gamma (from 4 to 1,000 pg/ml). Cells were then split and lysed
for Western blot analysis, or fixed and permeabilized for flow
cytometry. Percent of maximum (using 1,000pg/ml as maximum induction)
was calculated for all three analyses, and plotted versus interferon
gamma concentration. MFI (mean fluorescent intensity) values
were used for flow cytometric data, while integrated band intensities
were used for the Western blot. Note the strong correlation between
the flow and western data across all doses of interferon gamma. |
| |
| Krutzik,
P.O. and G.P. Nolan, Intracellular phospho-protein staining techniques
for flow cytometry: monitoring single cell signaling events.
Cytometry, 2003. 55A(2): p. 61-70. |
