2.4. Scanning electron microscopy

Fresh human whole blood was collected in Na-citrate tubes. Control or treated samples were centrifuged at 500g for 5 minutes followed by removal of plasma and buffy coat. Pelleted RBCs were washed twice with PBS and suspended in 0.1 M phosphate buffer at a hematocrit of 40 %. Next, RBCs in the phosphate buffer (pH=7.4) were fixed with 2.5% glutaraldehyde (GA) for 1 hour in room temperature (primary fixation). The suspension was then centrifuged at 500g and 5 minutes to collect RBCs which were re-suspended and incubated in 0.1M cacodylate buffer (pH=7.4) containing 1% osmium tetroxide for 1 hour in room temperature (secondary fixation). Thereafter, the RBCs were rinsed twice with cacodylate buffer and dehydrated serially in 30%, 50%, 70%, 85%, 95% and two times in 100% ethanol while adjusting the hematocrit to 20%. Finally, the samples were dried with hexamethyldisilazane (HMDS) followed by mounting and coating with palladium and examining using a Helios NanoLab™ 650 (FEI, Field Emission SEM).