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

Figure 3

Effect of Hb and NO on monocytes. Washed platelets isolated from healthy individuals were stimulated with Hb, collagen, or thrombin for 30 minutes at 37°C with gentle rotation and further incubated with DAF-FM-DA dye for measuring NO levels using flow cytometry. (a) Data represent from three different experiments showing the percentage of NO-positive platelets after incubation with the above agonists. , analyzed using a paired -test. (b) DAN assay was performed to quantify nitric oxide release from platelets. DAN (2,3-diaminonaphthalene) reacts with nitric oxide (NO) to form fluorescent napthotriazole (NAT), which was quantified by fluorescence spectroscopy with an excitation at 375 nm and emission at 450 nm. NO was quantified from the supernatant of platelets incubated with either Hb (3 μM) or thrombin (1 U/ml). Thrombin was used as a positive control. GSNO was used to prepare the standard curve for the quantification of NO release. Data are the from 3 experiments. and compared to unstimulated platelets. Hb (0 μM) was analyzed using one way analysis of variance with a Bonferroni post hoc test. (c) Monocytes isolated from the peripheral blood of healthy individuals were treated with either media alone, Hb, GSNO, or Hb+GSNO for 2, 24, and 48 h. After incubation, cells were harvested and processed for surface staining. Representative FACS plots from 3 independent experiments showing the percentage of CD14^dimCD16⁺ nonclassical monocytes (red gated). (d) A bar diagram showing the percentage of nonclassical monocyte subsets at different time points. Data are the from three different experiments. The statistical significance was calculated using a paired -test. and ; ns = nonsignificant.

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