Flow cytometric assay of pinocytosis: Correlation with membrane ruffling and metastatic potential in the dunning R‐3327 rat prostatic adenocarcinoma model

Abstract

Membrane ruffling has been associated with neoplastic transformation, Harvey ras expression, and metastatic capability. In the Dunning R-3327 rat prostatic adenocarcinoma model, membrane ruffling graded visually upon live cultured cells filmed by time-lapse video-microscopy has distinguished sublines of high and low metastatic potential. Fluid-phase pinocytosis is a constitutive, noninducible internalization of medium by cell membrane. Fluid phase pinocytosis may be measured flow cytometrically by cellular uptake of fluorescein-labelled medium constituents. The optimum conditions for a flow cytometric assay of pinocytosis were determined using AT-2 subline that has an intermediate degree of membrane ruffling. The optimum dextran concentration was selected from the midpoint of the linear portion of the dose-response (0.01-10.00 mg/ml) curve, whereas the optimum incubation time was determined from a time-course (1-405 min.) curve study. Cultured cells from 6 Dunning sublines incubated with 1.0 mg/ml of fluorescein-labelled dextran for 90 min were washed, fixed, and the fluorescence of 10,000 cells studied by flow cytometry. For each subline, dextran fluorescence was measured in four independent experiments. Pinocytosis failed to distinguish sublines of high (AT-3 63.5 +/- standard error 4.1 mean channel number, MAT-LyLu 63.2 +/- 6.3, MAT-Lu 64.3 +/- 5.6) and low (G 33.5 +/- 1.2, AT-1 63.5 +/- 4.1, AT-2 58.4 +/- 3.6) (rank p = 0.38) metastatic potential but correlated strongly with visually graded membrane ruffling (r = 0.95, p = 0.003). Pinocytosis assayed by flow cytometry reflects membrane ruffling observed visually and thus flow cytometric assays may facilitate study of membrane activity.