Cell Proliferation Assay.

Cell lines were analyzed for rate of proliferation. Proliferation was measured with the CCK-8 reagent (Sigma-Aldrich, MO, USA 96992). The strength of the CCK-8 reaction (bright orange color) is directly proportional with live cell number in the culture, therefore data was collected by measuring absorbance (optical density: O.D.) at 450 nm using a CLARIOstar® Microplate Reader (BMG Labtech, Ortenberg, Germany). PGC lines from the same breed were individually measured in the same experiment. The measurements were taken over 3 d, once per day. Triplicate wells were measured every day for every cell line on a 96-well plate (Lázár et al., 2018). Average absorbance values from each day was used to calculate doubling time (DT) for each cell line with the following formula: DT = d × log(2) / log(A_t) – log(A₀), where d: duration in days; A_t: absorbance at the third day; A₀: absorbance at the first day (Kong et al., 2018).