Hello,
I would like to treat cell culture with some drug for 12-24hr, then collect its protein sample to study cell signaling response. Should I collect the cell culture when it is still within exponential phase (no more than 70% confluence) or in stationary phase (80-90% confluence).
Thank you
Cheerio,
Obviously, it depends on the signaling cascade you are studying. As a "rule of thumb", I would collect lysate form exponentially growing cells, since cell-to-cell contact is known to strongly influence a number of signaling pathways. Maybe, for the first experiment, you could try both approaches (exponential and stationary phases) and check whether there are differences in your signaling of interest...
Good luck!
Stefano
I think you could collect both in the first experiment and watch the difference. It depends of millions of things, and every experiment is different.
Dear Asa
If you are determining signalling response to an apoptotic inducer confluency of cells will affect the outcome i.e. under less confluent conditions (30-50%) a greater rate of cell death and apoptotic signalling may occur vs 70% to 90% confluency. Likewise mitogenic signalling pathways have a more robust signal intensity in exponential vs stationary phase as cells in stationary phase often undergo G1 cell cycle arrest and senescence. I would advise you take these into consideration when designing your experiment and of course always use appropriate controls as far as possible.
Good luck with the experiments
:)
As suggested by others, I would do a pilot experiment with different cell confluences (simply start with different cell seeding number). As I have experiences, the response to the drug can vary greatly depending on the cell density (I am using HeLa, which are not as sensitive to cell density as other cells, but still show huge variation in response depending on the cell density). The I guess you have to decide your self based on what you are testing, which seeding density is est for you experiments. Good luck!
If you're seeding density is high then you probably want to start your experiment in exponential phase to avoid overcrowding. Also, it is going to depend on the plates that you are using. 24-well plates tend to get overly crowded in the center and makes it difficult for longer duration studies from what I have noticed with my own cell culture research.
It does not matter when they are collected, what is important is the cell density used for treatment, because as indicated by Esau, treatment response can depend on cell density, especially if its a signaling study where you have to look at live cells.
After deciding on density for the tissue culture vessel you are using, it is important to seed the cells and let them acclimatize for at least overnight before treatment. If very slow growing cells, you may have to culture longer before treatment.
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