I started working on cell line cytotoxicity testing for some of my compounds, mainly to determine IC50 on Hela cancer cells.
I have some question, please advice:
- I read that we should split cells (into T75), seed cells (to 96 well plates) when the cells are at 70-90% confluent. May i ask why 70-90? Above 90 may not good, but how about below? is that just because
I agree with the previous answer. In such conditions (70-90% confluence) you obtain active cells (you have to check it by blue assay for example). You are also able to obtain more reproducible results when these conditions are reached for all your assays. If other parameters are well managed the 70% - 90% had to be obtain at the same incubation time.
I think 70-90% is just a recommendation. I would say it would be fine to seed a lower density if you have the 96 well plates in culture before adding drugs.
It is important that cells are at the same confluency on treatment, however.
As a final point, we always find it useful to check area under the curve (kill curve) alongside IC/EC50. They each give a slightly different read out and having both match up can really show your reproducibility.
All above are correct but if you work on cancer cells, no need to worry about too much for the percentages given above as long as you are sure that your cells are proliferating succesfully. So, this means that if you have no time to wait for the proper confluency level (as given above), you may get your cells from a 50-60% confluent-T75 flask and seed them in a plate (or whatsoever) for cytotoxicity assay.
I think if you are not having good results with 70-90% confluency, you should try different confluencies and one concentration of drug that would be toxic to the cells.
Another way is to count how many cells per well you need in the 96-well plate then seed exatly the same number each time. It gives more reproducible results form one plate to another than diluting form a 70-70% conflulnt plate by eye
Thank you all, very much. You the researchers in this field are really very active.
May i ask one more question regarding PrestoBlue vs MTT. I think the adv of prestoblue is incubation time, it is really convinient while we only need 30 mins of incubation. So why MTT is still so popular?
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