Dear Sya Retno,

What do you want to know? If you want to know the percentage of cells in the S phase, then you need to label the cells impulsively for 30 minutes, for example. If you want to determine the percentage of cells in the G0 phase (that is, outside the cell cycle), then you need to incubate the cells for a time T (cell cycle duration) minus S (the duration of the synthetic phase of the cell cycle). Such a long doubling time of the cell population may indicate a significant proportion of cells in the G0 phase. Using my formula, you can calculate the duration of the cell cycle from the doubling time of the cell population and the percentage of cells in the G0 phase (see my review on the cell cycle)

Best regards,

Rustem Uzbekov

T =td / log 2 [(2 – y)/(1 –y)]

T - cell cycle duration

td - time of cell population duplication

y - part cells in G0 (non labelleld after long BrDU incubation)

Example from this paper:

1.1. Determination of cell cycle duration in XL2 cells. XL2 cells were seeded onto glasses, and after 48 h of cul
tivation their density was 1463 cells/mm 2 . After 72 h of experiment (120 h after cell seeding), cell density reached 7161 cells/mm 2 .

Thus, the number of cells increased by 4.89 times and average duplication time was 31.44 h (log2 4.89).

During this experiment, the proportion of cells in G0 was 0.179. Hence, average cell cycle duration was

T =31.44/log 2 [(2 – 0.179)/(1 – 0.179)] = 31.44/1.16 = 27.1 h (27 h 6 min).

Rustem E Uzbekov Prof Rustem, I want to check the cell cycle progression to my cell. I did a gene Knock-down based experiment and found out that KD cells have reduced proliferation rate compared to control. So, I was wondering if there is a cell-cycle arrest in this condition. My cell is a slow growing type of cells so I was just wondering how long I should incubate with BrdU

Dear Sya Retno,

In order to evaluate the change in the composition of the cell population in terms of the proportion of different phases, I can advise you to conduct a complex experiment. Cells of one passage are pulsed in parallel for 30 minutes with BrDU, this will give you the percentage of cells in the S phase (excluding cells in the G0 phase), in parallel, other cells of the same passage are incubated in the medium with

In order to evaluate the change in the composition of the cell population in terms of the proportion of different phases, I can advise you to conduct a complex experiment. Cells of one passage are pulsed in parallel for 30 minutes with BrDU, this will give you the percentage of cells in the S phase (excluding cells in the G0 phase), in parallel, other cells of the same passage are incubated in the medium with BrDU for 48 hours. The proportion of unlabeled cells will give you the percentage of cells in G0.

I would additionally suggest that cells from the same passage be pulse-labeled 30 min after 48 hours of culture in non-BRDU media to compare the percentage of cells in S phase at the beginning and end of the experiment.

At the same time, cells from the same passage can be counted over a small area in several fields of view N0 (mark with a marker on the outside of the culture flask). After 48 hours (t), count the cells in this area again (Nt). You will get the cell population doubling time td=t * Log2 (Nt/N0).

Alternatively, you can simply count 10 fields of view with an inverted microscope. But this may be less accurate due to some uneven planting of the cells in the flasks.

Further, according to my formula T=td/Log2 [(2-y)/(1-y)],

(y=part cells in G0 from 0 to 1) you can determine the duration of the cell cycle T from population doubling time td (using data on the proportion of cells in G0). In addition, knowing the percentage of cells in G0, you will be able to estimate, after recalculation, the real proportion of cells in the S phase (from the data of the first experiment). The percentage of cells in the G2 phase is determined by the method of labeled mitoses. Details are in my review and other articles.

The proportion of mitosis can be estimated directly with phase contrast inverted microscope observation.

The proportion of cells in G1 can be calculated by subtracting

G1= 1 - S - G0 - G2 - М

Finally, you can carry out all these experiments in parallel in control cells and in cells with a knockout of the studied genes. And get comparative data on cell cycle changes.

Best regards,

Rustem UZBEKOV

Excuse me, td=t / Log2 (Nt/N0)