I am doing MTT assay of TDP-43 transfected HEK293T cells in a 6 well plate. I am dissolving the MTT in DMEM media without Serum and Phenol red. After adding 850ul of the MTT solution to the cells I am incubating it in 37° incubator for 30 minutes. After 30 minutes I can see the formazon crystals forming in the medium but they don't precipitate properly in the bottom of the well, more like floating in the medium. After that without removing the medium I am adding 1.7 ml of DMSO to the cells. Do I need to remove the MTT medium before adding DMSO?? And if I remove the medium then would it result in loss of formazon crystals as they aren't precipitated properly in the bottom of the well??
Hello, Madhuparna!
The medium does not need to be removed before adding DMSO. In order for the crystals of pharmacan to settle to the bottom, it is necessary to incubate with MTT for 2.5-3.5 h, and not 30 minutes. After adding DMSO, it takes a very long time and carefully pipetting to break up the crystals
Hello Madhuparna
First and foremost, incubation with MTT for 30 mins is not enough. As mentioned by Alexandr Chernov you will have to incubate for a longer time in MTT so that the formazan crystals are formed completely.
Answering your question, yes, it is necessary to remove the MTT medium before adding DMSO because DMSO and the medium considerably changes the absorbance spectrum of formazan due to sodium bicarbonate present in the medium. Moreover, the color of MTT could interfere with the absorbance. The formazan crystals are soluble in DMSO and so there is no need of the medium while adding DMSO. You were right in using MTT in serum-free medium because formazan production is greatest in serum-free medium and decreases with increasing concentration of serum.
So you should remove MTT medium before adding DMSO for accurate O.D. readings.
Best Wishes.
Hello, Madhuparna
Although In some protocols it was mentioned that you can add DMSO without removing the MTT medium (Specially for non-adherent cells), based on the final results of my experiments I observed that it is necessary to remove the MTT medium before adding the DMSO.
It has been years since I've done MTT assays, but the kill solution I have always used for MTT assays is 4mM HCl/0.1% NP-40 in isopropyl alcohol. NP-40 lyses the cells and the dilute HCL in isopropanol dissolves the MTT so you get a uniform suspension. IIRC, I plated my cells in 100 microliters, added 25 microliters MTT solution, incubated 4 hours and killed with 100 microliters of the HCl/NP40/isopropanol solution, mixed with a multi-channel pipet, and read the OD.