Cx4 microscope

Five separate wells with cover slips were prepared for each oxygen condition (Norm, IH and SH, total 15 wells) at the re-plating step on day 3, and from day 3 to 7 an individual well in each condition was pulsed with bromodeoxyuridine (BrdU) (Sigma-Aldrich) at a 100 mmol concentration for 6 h, thereby providing a “snapshot” of the frequency of proliferation occurring for a 6 h period on each day in culture (10 (link)). After the last cycle on each day the supernatant was removed and colonies were fixed with ice-cold methanol for 10 min, washed with PBS and immersed in 2 M HCl solution for 2 h. Then the cells were washed 3 times with 0.1 M boric buffer and PBS before overnight incubation with anti-BrdU antibody (1:10) (Roche, Germany). Afterwards, the cells were stained with AEC staining mix kit (Sigma-Aldrich), washed with PBS and additionally stained with hemotoxylin, following by mounting with Immu-Mount mounting medium (Thermo, Pittsburg, PA, USA). Slides were analyzed with Olympus CX4 microscope at x10 magnification. Proliferation was expressed as the proportion of BrdU positive cells to EC-CFUs volume sum per well (cells/μm³). Colony volume (V) was calculated using the formula V = (πr²h)/3, where r = the radius of the base of the colony and h = the height of the colony, assumed to be equal to r (10 (link)).