If we want to check the effect of a small molecule on various cell lines of different sizes then which one we should keep same among them so that same amount of molecule will go in each cell among all cell lines? Cell number or total confluency? In my case, I am using RAW264.7, A549, MKN-1 and THP-1 cell lines. Thanks in advance.
Confluence as most cell lines display contact inhibition. Moreover the concentration of the small molecule is not affected by the cell numbers I assume
It depends entirely upon what you want to measure. Cell number is my preferred readout - it's simple, quantitative, and lets me know something about a molecule's cytotoxic and/or cytostatic nature. Confluence brings contact-inhibition into the mix, and is a bit harder to read out quantitatively. On the other hand, it tends to be a more selective readout for transformed cells (since they will often ignore some degree of contact inhibition when growing). Growth is soft agar is another common readout.
Ideally, do a good knockdown of your gene of interest, and pick an assay that has really strong, reproducible changes between the wildtype and knockdown cells. Then use that assay to look at your small molecules.
As for getting the same amount of molecule into each cell? That's a lost cause. Different surface areas, different expression levels for transporters, different membrane compositions - it's functionally impossible to scale your cell culture experiments such that exactly the same amount of molecule winds up in each cell between cell lines, so I wouldn't even worry about that aspect.
There is no way you can manage that same amount of drug enters all the cells. Also, if the cell size from various cell lines are different then cell number can’t be overall same in any case. You need to check literature for the different cell-lines, and optimize the assay in your lab.
This link gives seeding density and cells at confluency for HeLa cells and also mentions that the number of cells on a confluent plate will vary with cell type: https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
Cell physiology and response to toxins are affected by the cell density number; cells at a lower density are more affected by the toxin. Adversely, toxin may become bound and unavailable at higher cell densities.
The doubling time of different cells is different. Usually the control cells should be 70-80% confluent by the end of incubation period, and should not be over confluent in any case. Also generally, the greater the confluency, the lower is the toxicity.
Keep one factor constant and other variable, and optimize the assay. I would say both cell number and confluency are important.
Thank you Dr. Sven, Dr. William and Dr. Varun! I will keep the cell number same among all the mentioned cell lines at the time of treatment to check the effect of same molarity of drug on different cell lines.
- Badges
- Science method