Suppose I want to perform MTT of suspension cells. In which I seed cells in colourless media supplemented with 10 % FBS. Now I cant discard media while adding MTT. In that case FBS is present there while adding MTT. Is it causing any problem? I came to know that FBS interfere with MTT reading at 570 nm. Then what should be the procedure?
Hello Ananya,
For suspension cells you either
a. spin the 96 well plate at 1,000 x g, 4°C for 5 minutes in a microplate-compatible centrifuge and carefully aspirate the media.
OR
b. you add an equal volume of MTT solution to the existing media in culture. Ensure that the same volume of existing media is present for each sample.
You should add 50µL of serum-free media and 50µL of MTT solution into each well and incubate the plate at 37°C for 1-4 hours. In your case, since 10% FBS is present in culture media while adding MTT, you should use SDS-buffered DMF as solubilizing solution which would not require media removal but would need long period of time to efficiently dissolve the formazan crystals. If you use DMSO or isopropanol as solubilizing solution to dissolve the formazan crystals, these organic solvents might cause precipitation of proteins derived from the supplemented serum leading to the reduction in the sensitivity and accuracy of MTT assay.
Please find below the protocol to prepare an SDS buffered DMF solubilizing solution for MTT assay:
If SDS does precipitate, you may warm the solution to 37°C and mix to dissolve it.
Good Luck!
See: Analysis of Cell Viability by the MTT Assay
DOI: 10.1101/pdb.prot095505
- Badges
- Science topic