Hello, I am currently working on the 3T3 NRU phototoxicity test (OECD 432). However, during the 1hr 50 min incubation in EBSS buffer the cells are detaching from the microplate. Has anyone else had this issue at all? Can anyone offer any advice on how to prevent this please? Thanks in advance.
Why're you using EBSS? Do you incubate your cells in the incubator when the cell are exposed to NR in EBSS? Because EBSS is intended to short-term maintenance of cells at the presence of a CO 2 level environmental (0.04%, while in the incubator should be 5-10%). If you put the cell in the incubator the pH of the solution will drop and become acidic...you can use PBS instead.
If you leave your cell outside the incubator, then EBSS is OK, but, if this is the case, do you check the temperature?
Here's a protocol for NRU test
https://cellculture.altervista.org/neutral-red-dye-uptake-viability-test-nru/
hope this is helpful
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