Mitochondrial Morphology Analysis in Hematopoietic Stem Cells

MPPs or LT-HSCs were sorted into the slide coated with poly-l-lysine (SIGMA). Cells were placed on ice for 30 min to permit cells to settle onto the slide and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized with 0.2% TritonX-100 for 15 min at room temperature and blocked with 5% goat serum for 1 h at room temperature. Cells were incubated with the anti-p-H2A.X (Cell Signaling Technology, #9718, 1:200) or anti-Tom20 (Santa Cruz Biotechnology, sc-11415, 1:500) antibody overnight at 4 °C, washed and incubated in the Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, A11011, 1:1000) for 1 h at room temperature. Cell nuclei were stained with DAPI and mounted using fluorescence mounting medium (DAKO). Images were acquired with a Confocal Microscope A1R or super resolution microscope N-SIM (Nikon), and were processed with NIS-Elements software (Nikon). For quantitative analysis of mitochondrial morphology, images were first thresholded and then converted to binary images by using ImageJ software^{69 (link),70 (link)}. Individual particle (mitochondria) were analyzed for mitochondrial area and number of fragments.