Flow cytometric analysis of FITC-annexin V binding to RBCs. RBCs from normal (+/+) and band 3 null (−/−) mice were examined at the time of collection (0 hour, [A] and [C]) and after 36 hours (B and D) of incubation in PBS. The cells were washed three times in PBS before each measurement. (A) Shows the nonspecific background fluorescence of normal RBCs shortly after collection. Gate D defines the fluorescence intensity exceeding the level of nonspecific binding and is arbitrarily set to encompass less than 0.5% of normal RBCs. (C) Shows a subpopulation of band 3 null RBCs included within gate D (3.8% of the total population in the experiment shown; range, 3% to 5% in four independent experiments with increased FITC-annexin V binding. (B) Shows that normal RBCs show no increase in FITC-annexin V binding when examined 36 hours after collection. The subpopulation of erythrocytes included in gate D remains below 1%. In contrast, the percentage of band 3 null RBCs included in gate D increases to 15.7% at 36 hours (in the experiment shown; range, 15% to 20% in four independent experiments. Importantly, the entire population of band 3 null RBCs shows an increase in FITC-annexin V binding as reflected by the doubling of the mean fluorescence of the cell population included within gate F (entire cell population excluding cells in gate D), whereas no significant change is detected in normal cells. This experiment, which results are summarized in a tabular form, is representative of four independent experiments that yielded similar findings.