2.4. Cell Cycle Parameters

The cell cycle parameters in the regenerating tissue were assessed by the cumulative labeling method in experiments (2.1) and (2.2) at the stages of 1 to 3 dpa and 2 to 4 dpa, respectively. We incubated the regenerating worms in EdU solution for up to 48 h and fixed them after 15 min, 5, 10, 24, and 48 h of exposition. After EdU detection, we determined and plotted the LIs of each specimen at a certain time point, and fitted a cumulative curve for the obtained values. To evaluate the cell cycle parameters based on the cumulative curve that reached a plateau, we used a method described by Nowakowski and colleagues [50]. The cumulative curve is described by the equation: y = a + bx, where “a” is the intercept, “b” is the slope, meaning that before the break point, we observed a linear regression, then at the break point, the regression reached a plateau. After visualizing the cumulative curves in R by fitting them with the least squares method (with packages “nlraa” and “minpack.lm”), we estimated the approximate cell cycle parameters, such as the growth fraction (GF), the length of the S-phase (Ts), and the length of the cell cycle (Tc). The growth fraction was found by the plateau value of the curve when all dividing cells had an EdU label and the amount did not increase any more. The break point of the curve corresponded to the time Tc-Ts, so that by extrapolating a regression line to the y-axis, we found the Ts. Knowing the Ts, we could estimate the Tc by adding the Ts to the break point value. All of these parameters are described by equations from the mentioned model [50]:

and