Figure 7. Activity of iron reduction–dependent and –independent NTBI uptake pathways in classical monocytes.

Calcein-labeled PBMCs (n = 5 healthy individuals) were cultured in presence of 10 μM Fe³⁺ [Fe₂(SO₄)₃] with/without DFO (100 μM, A), L-ascorbate (100 μM, B), or BPS (100 μM, C) for the indicated time points. Monocyte subpopulations were defined as described in Supplemental Figure 2A. Calcein fluorescence in classical monocytes was measured by flow cytometry. Representative calcein signal histograms are shown (tinted histograms: Fe³⁺-only-stimulated cells, open histograms: costimulation). Graphs show ΔMFI values. Each point represents 1 measurement, bars denote mean, and error bars represent SEM. The cell donor is represented by symbol shape. Rate of calcein ΔMFI change was determined with second-order linear models. Estimates for calcein quenching rate in Fe³⁺-only-stimulated cells and differences in quenching rate between Fe³⁺-only and costimulated cells are shown with 95% CI. Estimate P values were calculated with 2-tailed t test. ANOVA statistics are presented in Supplemental Table 5.