Heme alters mitochondrial mass and dynamics. (A) Mt mass (i), Mt MP (ii), and Mt ROS (iii) monitored by flow cytometry in resident and recruited phagocytes of untreated and heme-treated (80 μmol/kg heme) Wt mice. Mt MP and ROS are shown normalized to Mt mass and expressed as MFI percentage or fold change over untreated Wt mice. Representative flow cytometry histograms of MTG staining of resident and recruited phagocytes are shown (i). (B) Representative confocal microscopy images of MTG-stained BMDMs untreated or treated with 5 μM heme-albumin (heme), alone or combined with 1 μM Pio or 50 ng/mL IL-4 for 14 hours, and relative Mt area and number quantification (i). Mt mass (ii), Mt MP (iii), Mt calcium (iv), and Mt ROS (v) monitored by flow cytometry in BMDMs untreated or treated with 5 μM heme-albumin (heme), alone or combined with 1 μM Pio for 14 hours. Mt MP, calcium, and ROS are shown normalized to Mt mass and expressed as MFI percentage or fold change over untreated BMDMs. Representative flow cytometry histograms of MTG staining of BMDMs are shown (ii). (C) mRNA levels of UCP2, DRP1 (i), CPT1a, ACOX1, VLCAD, and MCAD (ii) in BMDMs exposed to ACs (1 BMDM to 0.5 AC ratio) without or with 5 μM heme-albumin (heme) alone or combined with 1 μM Pio or 50 ng/mL IL-4 for 14 hours. mRNA levels are expressed in RQ. Data are average of 2 independent experiments. Values represent mean ± SEM. Statistical analysis was performed by comparing 3 or more groups with a 1-way ANOVA followed by a Bonferroni posttest. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.