Hello, I am testing a drug in order to see if it can stop the cell cycle. We sow 100,000 cells in a 100mm plate and treat it for 24 and 48 hours. The cells (tumoral and not tumoral) are not confluent but when we look at the cell cycle it appears stopped in G0 / G1, both the control and the treated one. We have been told that this is due to the confluence but they are not.
Why does this happen?
Thank you for your attention!
We are using several cell types such as colon and breast cancer (CT26, MDA MB231 and MCF-7) and also non-tumor as CCD18-CO, HEK ...When we sow them, we give them 24 hours to adhere and then we apply the treatment that we have other 24 and 48 hours.
In each time interval, we collect the cells and fix them with ethanol to later dye them with PI, therefore we perform a classical DNA content staining protocol of fixed cells.
Hi Azahara,
First, when I do this types of experiments I plan the pilot experiment for deciding the plating number of cells for each cell lines and each container. I plated serially diluted 7-8 different numbers of cells, which probably can located in the XY graph (X: days vs Y:cell numbers) at areas of lag phase, log phase and stationary (plateau) phase. I collect the cells after the certain days and count them with viability dye (PI or DAPI) without fixation. Then I choose the cell number between log and stationary phases at shoulder area. Even if the cells doesn't look confluent under the microscope every cell lines have a different sensitvity to their contact inhibition.
Second, you have to collect enough events ( I usually do more than 30,000 events from singlets for DNA cell cycle experiment) to get better statistics. Thus, you need to have at least 10 - 20 times more cells in control samples when you consider your cell loss at highest dose of killing by treatment.
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