Survival of PMos in SCD. (A) Representative IFC dot plot showing HD PMos gated on CFSE⁺ HO-1^hi and CFSE⁺ HO-1^low subpopulation in cocultures of monocyte/HMVEC/sickle RBCs. CFSE⁺HO-1^hi PMos and CFSE⁺ HO-1^low PMos were further analyzed and shown by representative IFC images and internalization index depicted by histograms. Left to right: single-channel BF, CD45 (representing PMo in purple), CFSE (representing RBC material), and merged images showing the position of CFSE⁺ materials with CD45⁺ cells. The internalization score was defined as in Figure 1D. (B) Representative dot plots comparing the frequencies of dead cells (ViViD⁺ cells) in PMos cultured with CFSE-labeled sickle RBCs and HMVECs in the presence of the HO-1 activity blocker SnPPIX or control (culture media). (C) The frequencies of dead PMos in HD PMo subpopulations of CFSE⁺ PMos (ie, RBC phagocytosed PMos) and CFSE⁻ PMos (ie, no RBC uptake) are shown as in panel B (n = 5). (D) Schematic representation of experimental design. CFSE-labeled RBCs were mixed with HD RBCs to mimic transfusion in patients with SCD and then cultured with HD monocytes and HMVECs. The uptake of CFSE⁺ RBCs by PMos was analyzed by FCM. Frequencies of CFSE⁺ PMos from HD (n = 5-10; E) and absolute number of dead CFSE⁺ PMos (n = 5-10; F) in the coculture as defined in panel D. (G) Frequencies of GPA⁺ circulating PMos (G) and percentage of PMos within total monocytes in patients with SCD (n = 12; H) before or after transfusion. Data represent mean ± SEM; means were compared using a 2-tailed Student t test. *P < .05; **P < .01; ***P < .001.