RBCs were then pelleted by centrifugation for 10 s at 16,100 g, washed once in LK or HK HBS to remove unbound LA-FITC and kept on ice until flow cytometry analysis. Unlike annexin-V, LA-FITC binds to PS in a Ca2+-independent manner and control experiments showed that binding was irreversible. Inhibitors were tested for self-fluorescence at their highest concentration with unlabelled RBCs.