I am going to do MTT assay on HK-2 (adherent cell line) cells? I have checked many different protocols online but they are so diverse and now I am not sure which one will be suitable for my cell line. I would be grateful if I could get some pointers. Thanks.
1) My supervisor told me to not count the cells and directly plate 300 ul of my cell suspension to 48 well plate.
2) After I have plated my cells and left it overnight for cells to attach to the surface of the plate. I will be removing the media next day from the wells.
3) I have prepared different concentrations of sodium oxalate in my media which I will add to my cells at different time points.
4) When its time to add MTT solution, do I need to remove the media and add MTT solution, wait for 4 hours, and add solevent, and read the plate?
So my doubts are as follows?
1) Is it ok to remove the media in Step 2 (above) and add my stimulant which I have dissolved in the media for my desired concentration? My stock concentration of Sodium Oxalate is prepared in water.
2) In step 4, according to MTT assay protocol that the company provided, it says to add MTT solution directly to the wells without having to remove the media.
Any suggestions and advice will be great. Many thanks.
Hi Ann I hope that my points will help you.
- We always change the medium before giving any treatment to the cells, so you are doing it right way.
- After your treatment time it is totally OK to add MTT reagent directly to the culture well there is no need to change the medium again.
- I recommend you the ATCC protocol for MTT https://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx
Also take note that MTT is inhibited by serum, so formazan might not form. Check if your media is serum free when you add MTT.
Hi Ann,
If it can help you, you can take a look at the protocol we've followed to perform a cell proliferation assay in a recent study ( Exploring tumourigenic potential of the parasite Anisakis: a pilot study. Parasitol Res. 2018 Oct;117(10):3127-3136. doi: 10.1007/s00436-018-6008-2. Epub 2018 Jul 13. PMID: 30006808).
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