Hi researchers
I have a problem
I'm doing a MTT assay on a ALL cell line. of course this cell line is suspended so I used u form 96 well Plates at first...
I seeded 20000 cells in 96 well plates and I incubated it 24 hours in incubator after 24 hours I centrifuge the plate with 1000 rpm in 5 minute after that I extracted my media and I treated my cells with my drug in different concentrations, so again incubation and after 24 hours I added 10 micro liter MTT solution, 4 hours incubation in incubator and again centrifuge... Again extracting the media in wells and at last I added 50 micro liter DMSO and rotating that in darkness, can you tell me why, after even 1 hour rotating, the crystals in treated well aren't solved?! What should we do now?! I can't read the OD with elisa reader!
It is so interesting that negative controls are completely solved😐
Hello
In MTT assay sometimes it is observed that there are extracellular formazan crystals formed which are much larger than the cells, and they grow longer over time. These growing crystals are marginally soluble and may begin to accumulate with seed crystals deposition.
I would suggest you add double the volume of DMSO (100ul) or change the solubilizing solution.You can use acidified isopropanol as the solubilizing solution.
I hope this is helpful.
Best.
I ran MTT assays in the past with suspension cell lines, such as JeKo-1, but after the MTT incubation, I terminated the reaction with 4mM HCl/0.1% NP-40 in isopropanol. I never had any problems being able to read the plates. I would suggest trying that.
I add 150 ul of DMSO and incubate the plate on a sahker at 37C for 40 min. It should help.
Best
@Malcolm Nobre
@Gary Lee Gilmore
@Daria Augustyniak
Thanks for all your help, I will check all your recommendations.
I would add to other responses that sometimes the plate may need really vigorous shaking (> 600 rpm).
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