Figure 3. Effect of heme on ROS formation in T. cruzi epimastigotes.

(A) Epimastigotes (1×10⁷ cells/mL) were incubated with 2 µM CMH₂DCFDA and different concentrations of heme. Images a, c, e,g and i show differential interference contrast, whereas b, d, f, h and j show fluorescence images. The CMH₂DCFDA (2 µM) signal indicated in green was acquired with λEm 517–527 nm: (a and b) autofluorescence, (c and d) no heme, (e and f) 30 µM heme, (g and h) 100 µM heme and (i and j) 300 µM heme. (B) Epimastigotes (1×10⁷cells/mL) were incubated in PBS with 2 µM CMH₂DCFDA and 30 µM heme for 30 min. The ROS formation was analysed by flow cytometry. The histograms correspond to: autofluorescence (gray), 2 µM CMH₂DCFDA (control-black), 30 µM heme (green), 100 µM heme (blue) and 300 µM heme (purple). The histograms are representative of five independent experiments. The inset graph represents the fluorescence intensity values obtained by the ratio of the experimental group median to the control group median (without heme). Data are expressed as the mean ± standard deviation (n = 5), * p<0.001 as compared to the control group by Tukey's test. (C) Epimastigotes (1×10⁷cells/mL) were incubated in PBS with 5 µM DHE and heme for 30 min, and ROS formation was measured by flow cytometry. The histograms show autofluorescence (gray), 5 µM DHE (control-black), 30 µM heme (green), 100 µM heme (blue) and 300 µM heme (purple). The histograms are representative of two independent experiments. The inset graph represents the fluorescence intensity values obtained by the ratio of the experimental group median to the control group median (without heme). Data are expressed as the mean ± standard deviation (n = 2), * p<0.001 as compared to the control group by Tukey's test. (D) Epimastigotes (1×10⁷cells/mL) were incubated in PBS with 1.25 µM Amplex red, 1 U/mL HRP and 30 µM heme for 30 min, and H₂O₂ production was measured in the supernatant by fluorescence spectrophotometry. Data are expressed as the mean ± standard deviation (n = 3), * p<0.01, ** p<0.05 and *** * p<0.001 as compared to the control group by Tukey's test.