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.