I have not encountered this problem before but for a period of 2 weeks I'm getting different optical density values when I read them just after 10 seconds. I agree with the concept of color density with addition of DMSO but I have checked on it before and I didn't get much variation in OD values within a minute. But I don't have any idea on what happened now. I would appreciate if anyone could let me know who faced the same problem and please give me your inputs.
you most wait 1-3 hours, to insure complete reaction of trypan blue by live cell line.
Hi,
yes, we had that problem, too, when using plain DMSO. There is a pH-dependent reaction of the MTT product even with low amounts of residual medium, leading to spectral change and lower absorption values, see this publication: Article A study of some variables in a tetrazolium dye (MTT) based a...
A simple solution to the problem is addition of a small amount of ammonia to DMSO, see here: Article Ammonia-containing dimethyl sulfoxide: An improved solvent f...
Also be sure to give the product enough time and agitation on a shaker to completely dissolve the formazan in the solvent, we generally use ~ 15 min.
you most wait 1-3 hours, to insure complete reaction of trypan blue by live cell line.
Trying to centrifugate the plate and put DMSO or HCL-isoproponol ( Acidic isopropanol is prepared as a 1.5% (v/v) solution of hydrochloric acid in isopropanol and stored in glass.)
Patrick Heinrich & Cássio Prinholato da Silva Your answers helped. Thank you for the reference too.
It is better to centrifuge the plate and remove the medium before adding DMSO and then the plate should be put on a shaker for a few min before taking readings
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