In my recent experiments to determine phosphorylated H2AX protein in response to adding a radio mimetic drug, I have been observing abnormally high levels of the the phosphorylated H2AX in the untreated cells, and samples of Untreated from early passage cells do not show such high levels of expression. So I was wondering if early or late passage and leaving cells that completely occupy the plate without passaging them for longer days (due to circumstances) might contribute to this abnormality. The cells I use are normal breast cancer cell lines.
If there is anything else that may cause the high background! That'll be very helpful.
If you keep cells at high density, you select for cells with mutations that abolish contact inhibition, that is, you select for a more transformed phenotype.
Hi Nivetha, do your confluent cells form a tight uniform monolayer or are there "dome" structures popping up from the monolayer?
Usually, as Annemarie said, many cells do not divide because they are contact inhibited. Cells that have lost that signal keep on dividing no matter what.
Sounds like you will have to determine what level of crowding or lack of crowding will not produce the high levels H2AX. Determining the quantities and rate of H2AX production in varying culture conditions might be a stand alone paper. Once you have those data, then you might see the rediomimetic drug effect for the second paper.
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