To generate stable mammalian cell lines, I am doing a kill curve to find the optimal concentration of antibiotic for selection. The problem I have is that, even with a very high level of antibiotic (~10X common literature values), a very low % of cells look kind of "okay" by light microscopy. Some protocols seem to indicate a low % survival is okay (eg, only 95% killing is required), other protocols say that you must kill 100% of cells.
My question is: What is the criteria for the "minimum" selection concentration for stable cell lines?
1. 100% of cells dead?
2. 95% of cells dead?
3. No growth of culture after a week?
4. Some other requirement?
Cells can often visually look okay by eye (or even by common counting methods like trypan blue excusion + hemacytometer) but actually be undergoing significant apoptosis/necrosis. I'd suggest that you set up a more comprehensive assay before settling on acceptance criteria. If you do this, I think you'll find that the answer might be more clear cut than it appears right now.
A couple of suggestions: if you have access to a flow cytometer, you can use a number of different viability dyes to check out how your cells are doing. Alternatively, you could use something like an MTT assay (Article Rapid Colorimetric Assay For Cellular Growth And Survival - ...
) as a colorimetric read-out of cell metabolism (you just need a basic plate reader for this).Good luck.
Hi Gen Gi
Checking by eye is seldom accurate, hence it is better to use an assay. My recommendation is Abcam's CCK-8 assay. Simply plate your cells into a 96-well plate with 100 ul of media containing different concentrations of media and incubate for an appropriate length of time, then add WST-8 reagent. This changes colour depending on metabolic activity. Finally, measure activity using a spectrophotometer at 460 nm.
https://www.abcam.com/cell-counting-kit-8-wst-8--cck8-ab228554.html
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line