I performed a flow cytometric analysis of apoptosis induced by synthesized compounds. i used 3 concentrations of single compound. each set incubated for 24h and 48h. i could easily figure out a dose dependent response at 24h. when analysis of 48h treated set were done, I could observe that the normal cell population were more than that of 24h treated cells.The preapoptotic cells and late apoptotic cells predominates in this set proving apoptosis. What could be the reason for increase in normal cells and how could i interpret the data?
I am assuming you are reffering to the percentage of parent population increasing at the 48hr time points from the 24 hour time point? Also, I am assuming you are using an Annexin V based assay?
If yes to all of those, one problem I had was making sure the number of cells being stained were similar for each time point. If there are double the number of cells at the 48hr time point as the 24hr time point, the mean fluor intensity could decrease and become included in the double negative gate if you are using the same gates as the 24hr time point. Just a pitfall I have walked into.
if that is not affecting your data, then I would wonder about the mechanism of your compounds. Are they phase specific (e.g. cytarabine in S-phase). If your cells cycle slowly, there could be a population of cells which have not been effectively disrupted by your compounds of interest. These are just variables to keep in mind
And if none of those seem to apply, and if your vehicle control seems to follow the same trend as your treatments, another thing I have noticed is seeding time. I have started to seed my cells at a lower density and allowed them to grow for 48hrs vs the typical 24 before I challenge them with any treatments. This helped to have consistent vehicle control data throughout the entire experiment (up to 72hr).
I hope some of my mistakes may help you,
Good luck
Hi - there is another explanation. It could be your compound is affecting a sub-set or sub-population of your cells and the other population(s) are unaffected (and proliferate over the later time points).
This may be especially true when using a cancer cell line (which often has cells of multiple sizes and ploidies present). If this is the case you may notice size distribution changes between the early (24 hr) and later treatment cells.
hope this helps
Gary
thank you otis william, tamara lekishvili and gary morley for your suggestions. I will clarify that i used HeLa cell line and Annexin V FITC/ Propidium iodide double staining..hope this will give a better idea..
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