To measure macropinocytosis, serum-starved A431 cells grown on coverslips and placed in Chamlid chambers were incubated with 0.5 mg/ml TMR-dextran and, where noted, stimulated with 100–200 ng/ml EGF in the indicated buffer for 10 min at 37°C. Cells were washed and both DIC and red fluorescence images of live cells were acquired. Where indicated, the following inhibitors were used: 10 µM latrunculin B, 100 µM LY294002, 1 mM amiloride, or 10 µM HOE-694. In the case of latrunculin B and LY294002 the cells were preincubated with the inhibitors at 37°C for 30 min before EGF addition. Macropinocytosis was quantified as the number of cells containing macropinosomes in the cells outlining each island.
Endocytosis was assessed by incubating the cells with 50 µg/ml Alexa 546–conjugated transferrin in the indicated buffer for 15 min at 37°C, after which the cells were placed on ice and acid washed with 0.2 M acetic acid in 150 mM NaCl and PBS to remove exofacial fluorescence. The cells were then fixed and mounted on slides, and red fluorescence was imaged and quantified.
Endocytosis was assessed by incubating the cells with 50 µg/ml Alexa 546–conjugated transferrin in the indicated buffer for 15 min at 37°C, after which the cells were placed on ice and acid washed with 0.2 M acetic acid in 150 mM NaCl and PBS to remove exofacial fluorescence. The cells were then fixed and mounted on slides, and red fluorescence was imaged and quantified.
