NSPCs were cultured in monolayer for five days until 80–90% confluency was attained, and passaged into three 60 mm PLO/fibronectin-coated petri dishes. At each passage, three plate cells were counted and viability assessments were performed using a Countess FL Automated Cell Counter (Thermo Fisher Scientific, Pittsburg, PA, USA) which is using trypan blue staining. The population doubling level (PDL), a cumulative counting technique, was applied using the following formula: PDL(n/n−1) = log (Nf/N0)/log 2, where n = passage number, Nf = final number of cells, and N0 = number of cells seeded at passage. To obtain the new PDL(n + 1), PDL(n/n − 1) was added to the previous PDL(n) [53 (link)].
For growth curve analysis, cells at the 3rd, 6th, and 9th passage were cultured in 24-well plates (coated 12-mm cover slips were inserted into each well). Cell counts were obtained daily for seven days using trypan blue to derive a weekly growth curve.
For growth curve analysis, cells at the 3rd, 6th, and 9th passage were cultured in 24-well plates (coated 12-mm cover slips were inserted into each well). Cell counts were obtained daily for seven days using trypan blue to derive a weekly growth curve.
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