Intermediate MKs exhibit high levels of mitochondrial superoxide anion. Mature MKs were collected after the BSA gradient and incubated for 5 (A-C; F-H) or 24 (D-E) hours before morphometric analysis by phase-contrast microscopy and Image J software and/or fluorescence analysis of individual cells after P-MLCSer¹⁹ or MitoSOX labeling. (A-E) Phase-contrast microscopy analysis differentiated 3 states of thrombopoiesis based on morphological parameters. (A) Representative images illustrate round, intermediate, and terminal MKs. Bars represent 10 µm. (B-E) The roundness index of the individual MKs was scored according to the equation (4π × area)/perimeter² at 5 (B-C) and 24 (D-E) hours. The number of cells was recorded according to the roundness index (B,D). The binning analysis (C,E) categorized the MKs into round (red), intermediate (blue), and terminal proplatelet (green) MKs, according to thresholds set at 0.40 and 0.83; dot plots show individual data and median lines (n = 3, Mann-Whitney U test). (F) Phospho-Ser¹⁹-MYL9 (P-MLC) fluorescence was measured in individual cells from the 3 populations of MKs by fluorescence microscopy and expressed as AUs normalized to the whole cell surface. Dot plots show individual data and median lines (n = 3, Mann-Whitney U test). ***P < .001. (G) MitoSOX fluorescence was measured in individual cells from the 3 populations of MKs and expressed as AUs normalized to the cell surface (n = 3; Mann-Whitney U test). *P < .05; ***P < .001. (H) Distribution of the 3 indicated populations of MKs after MitoTEMPO (10 µM) treatment; data are expressed relative to total cells (means ± SEM; n = 3; treated vs control cells ratio paired Student t test). *P < .05; **P < .01.