Fluorescence microscopy analysis of FITC-annexin V binding to RBCs. RBCs were examined after 36 hours of incubation in PBS to facilitate the detection of abnormal cells due to progressive loss of phospholipid asymmetry. Washed RBCs were incubated with FITC-annexin V at room temperature. After two washings in PBS, RBCs were collected by centrifugation at 1,000g and placed between siliconized slide and coverslip. Normal and band 3 null RBCs were examined by phase contrast microscopy (A and B) and fluorescence microscopy (C and D). Note that normal RBCs do not bind FITC-annexin, whereas some band 3 null RBCs show a conspicuous membrane-associated fluorescence reflecting FITCannexin V binding.