I am doing a colony formation assay on 96 well plates. Each well has 150 NTERA-D2 cells. The incubation times for this assay are 4 days, 7 days, 10 days, and 14 days.
I replenish the media every 4 days. However, on day 8 I noticed that when I replenished the media in the untreated negative controls, some of the cells in the center of the colony had detached or died (please refer to the photos to know exactly what I mean). When I observed the cells again on day 10, the cells in the control looked dead (shrunken and spherical).
I am very gentle when I replace the media in the wells, so I do not know why they are getting detached or dying. Is there anything I could do to stop my cells from dying?
The passage number of the cells was 18.
Using a 24-well plate or a 12-well plate is not an option either.
I have attached a photo of what healthy colonies should look like as a reference.
Any help is greatly appreciated.
Do you have any contamination? My cells start detaching and looking like this if they are contaminated.
Hi, from my experience if your cells have contact inhibition then cells in the center of the clone do not grow anymore and they are old so... they died . maybe you should read your assay earlier... also a guess; if water from the medium evaporates then you add new medium that evaporate also ... so you are slowly concentrating the medium maybe your cell do not like that
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