in MTT assay we use DMSO as solvent ,, but for how long? and is it necessary to be kept in dark place ?
Hello Nujud Saleh
Using DMSO as the solubilizing agent in MTT assay, incubate the plate for up to 15-20 mins at room temperature in the dark. You may wrap the plate in foil and shake on an orbital shaker (very low speed) for the required time.
Best.
Nujud Saleh
You can determine the optimal time by observing the solubilization of formed formazan. 37 C can accelerate the solubilization and RT is also fine.
Hello, Nujud Saleh
After adding DMSO to solubilise the formazan, incubate it at a set time (usually within 15 minutes) in the dark or wrapped in foil. Long-term exposure of MTT to light may increase the formation of formazan - may cause variance in overall results if it is taken lightly.
A few common ways to solubilize the formazan:
For consistency, I personally set on the protocol for all my plates. Away from direct light, add DMSO and solubilise formazan completely, 5 minutes incubation and immediately obtain absorbance.
Hope this helps. All the best!
Hi Nujud Saleh
Post adding DMSO, Keep the plate in shaker at 10-15rpm for 15mins. That should be sufficient to solubilize the formazan crystals and take absorbance reading at 570nm.
The absorbance value obtained from each well is proportional to the number of viable cells, and you can compare the absorbance values between different experimental conditions to assess cell viability and proliferation.
Thanks,
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