(A) Flow cytometric analysis of RBC microvesicles shed from stored RBC. Two units of packed RBC units, stored for 35 days at 4°C, were washed with PBS. RBC microvesicles were gated by forward scatter/side scatter to include microparticles, smaller than RBCs, staining with antiglycophorin; scattergrams with gates can be seen on the right. Gated microvesicles of the two units (above and below) stained with CD59 and antiglycophorin are demonstrated on the left. (B and C) Immunoblot of RBC eluate, microvesicles, and HDL. Samples of RBC eluates (1), microvesicles (2), and HDL (3) were solubilized with 3% SDS and analyzed by immunoblot (on 12% polyacrylamide gel) with CD55 (B), CD59 (C) MoAbs. Bands at 19 (B) and 60 kD (C) correspond to CD59 and CD55, respectively. (D) Immunoblots of four commercial HDL preparations (labeled 1 through 4) stained with CD59 MoAb (on 14% polyacylamide gel). A band at 19 kD corresponds to CD59.