Figure 3
Flow cytometry of 293F cells and titin-transfected cells. (a) Gating strategy of 293F cells and titin-transfected cells. Cells were incubated without serum and secondary antibody. The gating strategy involved first identifying the 293F cell population by forward scatter (FSC) and side scatter (SSC) (left, upper). Live 293F cells were gated and measured fluorescent intensity of GFP and PE to define GFP-negative 293F cells (middle, upper). In addition, PE fluorescent intensity of GFP-negative 293 cells were analyzed by histogram plot (right, upper). Live titin-transfected cells were also gated by SSC versus FSC density plot (left, lower). We defined GFP-positive titin-transfected cells by PE versus GFP fluorescent intensity density plot (middle, lower). PE fluorescent intensity of titin-transfected cells were analyzed by histogram plot (right, lower). (b) Background PE fluorescent intensity was defined. Cells were incubated without serum and with secondary antibody. GFP-negative 293F cells and GFP-positive titin-transfected cells were gated. We defined background PE fluorescent intensity as shown in histogram plot (right, upper and right, lower). The proportion of background PE-positive cells was calculated as 0.5%. (c) The PE fluorescent intensity of titin-negative serum was analyzed. Cells were incubated with titin-negative serum and secondary antibody. The proportion of PE-positive/GFP-negative 293F cells and PE-positive/GFP-positive titin-transfected cells of titin-negative serum was calculated as 1.61% (right, upper) and 0.91% (right, lower), respectively. (d) The PE fluorescent intensity of titin-positive serum was analyzed. Cells were incubated with titin-positive serum and secondary antibody. The proportion of PE-positive/GFP-negative 293F cells and PE-positive/GFP-positive titin-transfected cells of titin-positive serum was calculated as 5.76% (right, upper) and 93.53% (right, lower), respectively. SSC = side scatter; PE = phycoerythrin; FSC = forward scatter, GFP = green fluorescent protein; PBS = phosphate-buffered saline.
