Figure 4.

FACS analysis with membrane-impermeant E2 and anti-ER antibody. (A) HUVEC were harvested by nonenzymatic cell dissociation and single-cell suspensions of nonpermeabilized cells stained with E2coBSA-FITC and anti-ERα mAb 1D5 plus GAM-PE in the presence of 5% BSA. Background gating of cells stained with FITC and GAM-PE alone was performed as a negative control (99% of cells within left lower quadrant, data not shown). Five thousand cells were analyzed per sample. FL1-Height: FITC fluorescence relative to gated background; FL2-Height: PE fluorescence relative to gated background. Statistical analysis of quadrants is shown. (B) EA.hy926 monolayer cells were harvested and single-cell suspensions of nonpermeabilized cells stained with E2coBSA-FITC in the presence of 5% BSA. Indicated samples were treated on ice with ICI 182,780 or a 20-fold excess of E2 for 1 h before E2coBSA-FITC staining. Five thousand cytometer-acquired events were analyzed per sample.