I need a general protocol for the assay. How long should I incubate microglia with the tested compound?
I have tried incubating the cells for 24 hrs with the drug, but the problem I have is that the absorbance of the blank (DMEM culture medium without any cells) is always greater than the absorbance of the samples containing cells. This is very strange because the absorbance produced by cells should be significantly higher than that produced by DMEM medium alone.
I have seeded 50,000 cells per well in a 96 well plate and incubated these for 24 hrs with the drug. Then I removed the drug containing the medium and added fresh medium (100 ul per well) together with 10 ul of WST-1 reagent per well and incubated the cells for another 24 hrs. Then I measured the absorbance of the samples and to my surprise the absorbance of the blank (DMEM alone) was greater than the absorbance of all samples containing cells. Has anyone had a similar problem? Do you have any suggestions?
Hint: I use primary microglial cell cultures that are freshly isolated from mouse brain tissue
Hi, did you find the reason why absorbance is highest in blank samples? I have the same problem and would be appreciated for any suggestions....
Unfortunately, I didnt find an answer and I had to stop these experiments, but I would recommend if you try the lactate dehydrogenase release test as an alternative. Otherwise, you may need to make a time-dependent experiment where u try out different incubation periods and see which one would give u the optimal results. In my experiments, I only tried out the 24 h incubation period
I think the problem is about the measurement but not the incubation process, because i actually treat cell with a chemical for just 30 min and this time is enough for my experiment. I just want to see if this incubation kills the cells or not. However thank you for the suggestions.
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