*4.4. Immunocytochemistry*

For immunocytochemistry, hCPCs were seeded at a density of 50,000 cells per well in a 2-well chamber slide (BioTek, Winooski, VT, USA). After pretreatment with histochrome for 24 h, cells were washed once with PBS and fixed in 4% paraformaldehyde (PFA) for 10 min. Cells were then permeabilized in 0.1% Triton X-100 + 0.01 M Glycine + Phosphate-buffered saline (PBS) solution for 30 min. Slides were blocked with 10% normal goat serum in PBST (0.1% Triton X-100 in PBS) and incubated for 1 h at room temperature. γH2A.X (phospho S139) antibody (#ab11174, 1:500 dilute in blocking buffer, Abcam, Cambridge, UK) was diluted in blocking solution and incubated overnight at 4 °C. The following day, slides were washed 3 times with PBS and incubated with Alexa Flour 488 goat IgG anti-rabbit antibody (1:200, Invitrogen, Carlsbad, CA, USA), and incubated for 1 hour in the dark. Following cell wash twice, cells were mounted using ProLong diamond anti-fade mountant with 4 ,6-diamidino-2-phenylindole (DAPI). Slides were analyzed under a Lionheart FX automated microscope (BioTek, Winooski, VT, USA)
