Fig. 2. Flow cytofluorometric analysis of phalloidin staining of RGM1 cells as a function of the presence or absence of Ca2+ during wounding. (a) Cells were wounded by scraping them from the substratum or incubated undisturbed in FDx for an equivalent interval. Flow cytometry was then used to measure the fluorescence of 10,000 cells in the undisturbed (gray field) and scraped (bold trace) populations. Greater than 92% of the scraped (W, demarcated by right arrow) population fell above a fluorescence threshold set to contain 95% of the undisturbed population (NW, demarcated by left arrow), indicating that this percentage incurred plasma membrane disruptions as a result of scraping. (b) Cells were scraped from the substratum in PBS containing 1.5 mM Ca2+ (Ca2+; bold trace) or containing no added Ca2+ and 1.0 mM EGTA (EGTA; gray filled). Flow cytometry was used to measure the fluorescence of 10,000 cells in each population. (c) The mean (and s.d.) of the FITC-phalloidin fluorescence measured by flow cytofluorometry from 4 populations scraped separately from four dishes, in EGTA (EGTA)- or Ca2+ (Ca2+)-containing medium. Applying the Kruskal-Wallis test to these two data sets gave P<0.05.
