I observed this phenomena with different cell lines and anticancer agents. To be specific for example, when A549 cells were treated with same concentration of taxol, less cells die if the cells were seeded in a 25cm2 or 75 cm2 flask compared to the cells seeded into 96 well plates (microscopic observation). Ratio of the seeded cell number to the surface area was the same, but the result did not change. Does anyone have an idea what might cause this?
Rather than a microscopic observation I would do a cell based assay, I use MTS in 96 well plates and cell counts for flasks then work out the percent cell death relative to untreated cells. If you are seeing a real difference then it may be that the vessels have different surface coatings. I would try to use either plates or flasks rather than both if possible in order to ensure consistency in your data.
Dear Mehmet Sarimahmut,
I had hard troubles on cell growth and morphology too. I usually work with HTB-126 and HTB-125 cell lines. If you buy cell from come repositories you should follow carefully what the cell's data sheets suggest you to do, concerning cell culture.I had trouble to grow up cells number in flask instead of dishes. Usually repository suggests which cell dish or flask is suitable for the cell line you're working with. Did you plate the cell at the different concentration for each reservoir but you used the same drug concentration?If it so, this might be affected your results. You used the same drug concentration for a different cell concentration. In this case less cell number cannot face out drug as well as higher cell number. I hope my reply can help you :-)
Bye for now :-)
In my experience 96 well plates are not the best option for assaying cell growth and i try to avoid them as much as possible. Ultimately it all depends on your specific cell line, but i'm not familiar with the A549. Generally cells that are round-shaped and grow in monolayers perform better in 96 well plates compared to metastatic cells that are more stellar-shaped and grow in clumps. Ultimately surface available for growth is reduced in a small well compared to a flask, so cells will behave differently and this will impact on your chemotoxicity assay in 3 ways:
1) provided same initial seeding density and same drug concentration, cell density per square mm will be different and this will impact on cell surface available for drug interaction (although this obviously depends on the kind of drug you are using, wether it acts on the cell membrane or needs to be taken up by the cell)
2) Higher cell density means more cell-contact inhibition, which ultimately also impacts on cell signaling and cell behavior.
3) Cells that grow faster also reach confluency very quickly in a small well, making it more difficult to appreciate differences with drugs that act with a slow onset.
If you are using MTT is obviously more expensive to scale up your experiment to a 48 or 24 well plate, but you can use alternative methods such as the neutral red assay (http://www.nature.com/nprot/journal/v3/n7/full/nprot.2008.75.html) which performs as good as the MTT but can be scaled to larger vessels without compromising on cost (i do it regularly on 24-well plates).
Dear Luca, the number of seeded cells was constant per mm2 area. So the density was the same.
Thanks for all replies.
It is the quality of plasticware used that makes difference at times. Occasionally a particular lot even from a reputed brand does not give consistent results. It is necessary to rule out the margin/periphery/border effect seen in 96-well plates.
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