PMo uptake sickle RBCs when cocultured with HMVEC. (A) Schematic representation of experimental design. HMVECs were pretreated with hemin or PBS for 2 hours and subjected to 2 washes before the addition of purified total monocytes and CFSE-labeled RBCs. Some monocytes and labeled RBCs were cultured in the absence of HMVECs. After overnight cultures, the uptake of CFSE⁺ RBCs by monocyte subsets was analyzed by FCM and IFC. (B) Representative dot plots comparing CFSE⁺ PMos and CFSE⁺ CMos from cultures shown in panel A. Frequencies of CFSE⁺ monocyte subsets from HDs (n = 3-12; C) and patients with SCD (n = 6-12; D). (E) HMVECs were preincubated with individual blocking antibodies (anti-ICAM-1, anti-VCAM-1, anti-CD11a, anti-CD11b, anti-P-selectin, anti-PSGL-1, anti-CD11c, anti-CD18, anti-CD31, anti-CD16, anti-CD32, anti-CD64, anti-Fc α/µ receptor, or isotype matched control [mouse IgG] antibodies) or annexin V or tin protoporphyrin IX (SnPPIX) for 30 minutes before the addition of HD monocytes and CFSE-labeled sickle RBCs. It should be noted that it takes ∼10 to 15 minutes for monocytes and RBCs to settle on the HMVEC. Frequencies of CFSE⁺ PMos were then analyzed (n = 4-12). Data represent mean ± SEM; means were compared using a 2-tailed Student t test. *P < .05; ***P < .001. Ab, antibody.