Please contribute to this question with your assured knowledge.
In treating cells for different time spans like 24h, 48h and 72h, should we seed different numbers of the cells suitable for relevant treating time?
If yes, what is the method to define the best seed number?
If not, in the case the cells continue to grow more in higher treatment times (though the viability decrease as drug concentrations increase), does higher treatment time worth to continue studying?
Thanks for your participation.
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The above may be helpful to start with.
Hi Arash Azimzadeh-Isfanjani
In my opinion you should keep the same cell numbers for each time point. Each experiment should be simple and only test one parameter, but you can run different experiments to test different parameters.
For example, you could first determine a suitable number of cells to seed for 24h, 48h and 72h - this will be a concentration that cell growth continues, even at 72h. Seed too many cells and they will begin to die before 72h. Seed too few cells and there will be limited change in growth. For a 96-well plate, I would recommend about 15,000-40,000 depending on the division time for your cell line. This is your preliminary experiment to determine the most suitable number of cells.
Next, you can seed the most suitable number of cells into your wells and perform your experiment with treatment over 24h, 48h and 72h. Finally, you can run a slightly different experiment where you plate different cell numbers for a particular length of time (24h for example) to determine how cell concentration affects your treatment.
Hope this helps and good luck!
Basically you are optimising two things:
1. Cell number
2. Concentration of drug
First optimise your cell number per well with known stimulant..check for its metabolic activity using real time MT Glo kinetic study..or by some other methods..then optimise the concentration of drug using definite cell number per well
Hello Arash Azimzadeh-Isfanjani
You need to seed cells at the same density for various time points so that you can keep variations in the assay to the minimum. You can seed cells at a lower density so that they do not become over confluent at the 72-hour time point. 70-80% confluency is sufficient for the assay to check the action of the candidate drug. You may carry out a pilot experiment to optimize the seeding density for the assay.
Longer treatment time is necessary in certain cases because some cells may not respond to the drug at lower incubation time point. In such a case, you would need a higher incubation time to see the drug effect on the cells. For instance, in MTT assay, three time points are considered namely, 24,48 and 72 hrs. At times, depending upon the cell type and the properties of the candidate drug, you may see a marked cytotoxic effect after 24 hours or it may even prolong up to 72 hours.
So, the initial cell number which has to be the same, and the higher treatment time is worth considering.
Hope this helps.
Best.
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