Doubling-time assay

Detailed Cell Doubling Time Assay:

MCF10a cells were cultured as described in the methods of Sato et al 2021 

Prepare 200 uM 4-hydroxytampxifen (4OHT) diluted in 100% Ethanol (EtOH) ahead of time 

Cell preparation:
Collect exponentially growing MCF10a cells via trypsinization, followed by centrifugation and resuspension in growth media. Ascertain cell concentration via the Countess II automated cell counter. 

Dilute cells in growth media to a final concentration of 2.25e³ cells per ml. Aliquot two 13ml volumes of the starter stock 

To one aliquot add 26ul of 200uM 4OHT for a final concentration of 400nM. For a vehicle control, add 26ul of 100% EtOH to the other aliquot. 

Dispense the drugged aliquots in a 6 well tray, 2 ml of stock preparation per well for a final concentration of 4.5e³ cell per well. This concentration will ensure logarithmic growth throughout the course of the experiment. Seed 6 wells for the +4OHT condition and 6 wells for the vehicle control condition

Allow the cells to grow for 72 hours (3 days) before collecting the first time point 

Cell collection and counting: 

Collect three of the 6 well for each condition by trypsinization for three technical replicates. Take note of precise time of cell collection, and identify a time post seeding in hours (T₃). Centrifuge the samples and resuspend in 100ul of growth media. Count the cell concentration via countess II in duplicate for each replicate and average to gauge cell concentration. Then calculate cell yield for each replicate by dividing concentration value by ten. Average across the three technical replicates to calculate day 3 cell yield (Y₃)

On day 6 (72 hours post day 3 counting, 144 hours post cell seeding) repeat the cell collection steps described above to calculate day 6 cell yield (Y₆) and a time post seeding (T₆)

Calculations: 

Number of doublings: To calculate the number of doublings that occurred between day 3 and day 6, take the base 10 log of each cell yield value, and subtract log(Y₃) from log (Y₆). Divide this value by log(2) to calculate the number of doublings that occurred between day 3 and day 6 

Doubling Time: determine the time between the cell counts by subtracting T₃ from T_6, then divide this value by the number of doublings that occurred during that time. This will give you the time per doubling, or doubling time value 

Statistical analysis 

Each experiment will only yield a single doubling time value for each condition, so the experiment should be repeated three times for three biological replicates to allow statistical testing.