Macropinocytosis assay

KPC cells in complete DMEM were seeded into the wells of 24-well culture dishes containing circular coverslips that had been washed with 95% ethanol, and allowed to settle and grow overnight. When the cells reached ~65% confluency, media was removed and cells were incubated with serum-free media for 2-4 h, then with serum-free media containing 0.1 mg/ml Tetramethylrhodamine (TMR)-Dextran and incubated at 37°C for 30 minutes. Plates were placed on ice and gently washed 5-7 times with ~2 ml/well cold PBS. Cells were fixed with 3.7% (v/v) formaldehyde solution in PBS at room temperature for 15 mins, then rinsed 3 times with PBS. Cells were consecutively stained with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific) and then DAPI (0.15 µg/ml). Coverslips were affixed to glass slides with DAKO mounting medium and stored on a flat surface in the dark overnight prior to imaging using an Olympus FV1000 microscope. The macropinocytic index was quantified as described in (26).