I want to set my protocol to do mtt assay of peptide and protein. I have been suggested on medium that should contain FBS, or on medium that has to be free from FBS for incubation of peptide drug with cell.
One group is saying that medium containing peptide for incubation with cell, should not contain the FBS (Fetal Bovine serum ). Incubate cell in these medium with peptide drug for 4 hours and then change the cell medium with medium containing FBS. Incubate requisite time and then add mtt to check cyototoxicity.
Another expert group is saying to use the complete medium (with FBS ) containing peptide. First incubate the cell in these medium for requisite time and then add mtt to check cytotoxicity.
Both groups agree on the point, that for non-peptide drug, one should use the complete medium ( with FBS ) and drug, incubate for requisite time, and add mtt to check cytotoxicity.
Can someone solve the anomality with the peptide containing drugs?
Hi
I agree with Shiv. You should growth the cell in complete media (+FBS) and prepare you test sample (dilutes sample) with the same media for cell growing. After incubate for requisite time, remove media in the cell growing plate and replace with fresh media that has test sample was prepared. Incubate for requisite time and add MTT to check cytotoxicity ability.
Thank you shiv kumar and Trung,
But I have another query, is it possible that serum in the media may decrease the activity of peptide and protein as the serum has binding property with most of the peptide and protein and decrease their bio-availibility.??
Do both, if the percentage change determined by MTT is different then your FBS is having an effect. Of course not having FBS is likely to affect the overall number of cells at the end of the incubation period. I used to establish cells in culture with FBS media then remove from incubation media as Trung suggested.
Just to reinforce the suggestion from Dr. Butler: there are several reports on the impact of the presence of even small amounts of FBS during the incubation with MTT. Even international standards for cytotoxicity tests, such as ISO 10993-5 recommend the incubation in absence of FBS. In my lab, we also establish the culture in presence of protein, then change the medium for the cell assay.
It could be also interesting to consider other tests with less interference from proteins, such as Neutral Red....
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