Hi folks！ I am currently doing microplate fluorescence screening for cell viability test. Some friends of mine told me that there would be interference when I put well plate with cells inside directly to the reader. They said cells would somehow block the light or something like that. But according to my experience, the readout is significantly different if I increase the drug dose. So I think it is fine. I just want to know how serious the interference is, and how that could happen.
Hi kenny
The cells would indeed interfere however minimal and it varies from one wavelength to another; that is why it is important to make a standard curve from those cells with different cell density without your test (as a bench mark; aside from your control), then read your absorbance with the tested cells (and control) and relate it to your standard curve.
I know it is lengthy procedure and there are other ways to read the cell viability without standard curve using calculations (https://www.bio-rad-antibodies.com/measuring-cytotoxicity-proliferation-spectrophotometry-fluorescence-alamarblue.html) however this method has its own flaws; like an increase in absorbance against control may just be because of a 1000 cells or a 100,000 cells you might not know that without the standard curve.
Hi Kenny, based on my personal experience, I think it's OK to read 96-well plate containing cells directly using a microplate reader. I agree with Saif that you need to optimize the conditions for your assays, for example, how many cells you need to seed per well to meet the reading range of the instrument. A standard curve is probably needed.
Hope it's helpful for you!
Thanks, Saif! Very helpful of the explanation. But I actually think even Alamar Blue still requires standard curve for calibration? I am actually using this dye for absorbance test, lol. I think I know what to do, thanks again.
Thank you, Han! Because I have been doing the test for a while, data were all collected based on cells-containing reading. very much worried... Now you answer really soothes me~
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