Absence of Nonclassical Monocytes in Hemolytic Patients: Free Hb and NO-Mediated Mechanism

Figure 4

Hb and NO mediate the death of nonclassical monocytes. Total monocytes or separately nonclassical and classical monocytes from healthy individuals were incubated with media alone, Hb, Hb+collagen, Hb+thrombospondin, and Hb+GSNO for 2, 24, and 48 h. After incubation, cells were harvested and processed for 7-AAD staining to assess cell death using flow cytometry. (a) Representative FACS plots from 3 independent experiments showing the percentage of 7-AAD-positive monocytes. (b) A bar diagram showing the percentage of 7-AAD-positive monocytes at different time points. Data are the from three different experiments. The statistical significance was calculated using a paired -test. and ; ns = nonsignificant. (c) FACS sorting strategy of monocyte subsets from PBMCs. Cells were gated based on size (FSC-A vs. SSC-A), and doublet discrimination (FSC-H vs. FSC-A) was performed. Monocytes were gated as CD14⁺CD11c⁺ and further sorted based on CD14 and CD16 markers. After incubation with different treatments, monocytes were processed for 7-AAD staining. FACS plots (d and f) and bar diagram (e and g) showing the percentage of 7-AAD-positive nonclassical (d and e) and classical (f and g) monocytes, respectively. Data are the from three different experiments. The statistical significance was calculated using a paired -test. , , and ; ns = nonsignificant. Hb+GSNO treatment to nonclassical monocytes significantly increased cell death.

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