Mitochondrial ROS and mitochondrial biogenesis in CLL cells. (A) Mitochondrial-specific ROS production was detected by the MitoSOX probe and was semiquantified based on the MFI of MitoSOX by FACS in HD B-cells (n = 5) and CLL cells (n = 5). (B) (left panels) 2 representative histograms of a FACS analysis of CLL cells stained with MitoSOX (for mitochondrial ROS) and CellROX (for total cellular ROS) with (black line)/without (filled gray) MitoQ treatment. The mean increase/decrease of the MitoSOX- and CellROX-MFI is shown for CLL cells (n = 5) treated with MitoQ or diphenylen iodonium (DPI) blocking NOX activity. (C) Mitochondrial mass was visualized using Mitotracker (green) in CD19⁺ (red, encircled) B-cells and CLL cells by fluorescence microscopy as shown for 2 HDs and CLL patients, respectively. Samples were counterstained with the vital nuclear dye Hoechst (blue). (D-E) The 2 panels display the semiquantification of the mitochondrial mass in normal B-cells (n = 6-10) and CLL cells (n = 6-10) based on the CLL cell/B-cell quotient (in %) of the Mitotracker-MFI (left panel) and the relative (to nuclear DNA) mitochondrial DNA (mtDNA) copy number (right panel). (F) A representative electron-microscopic depiction of the increased number of mitochondria (red circles) in B-cells and CLL cells. The mean number of mitochondria per B-cell and CLL cell is shown as a dot plot. Thirty cells per donor (n = 3 for each cohort) were analyzed using electron microscopy. (G) Total cellular ROS as determined based on the CellROX MFI in CLL cells measured by FACS was correlated with the according mtDNA content as quantified by real time PCR. Bars indicate the standard error mean. Abbreviations: P, P-value; *P < .05; **P < .005; ***P < .001.