I am performing MTT of ZnONPs on MCF-7 cells. I have come across two different protocols where, on Day 2 treatment with NPs
1. 100ul of NP dilution prepared in incomplete media is added to wells with complete media 100ul of seeding day containing cells. And even after treatment, MTT addition is also done in that 200ul of media only. So basically, no removal of media step anyday.
2. On day 2 remove the complete media containing FBS, then add 100ul of NP dilution prepared in incomplete media to wells. Next day remove the media completely and then add 100ul of MTT to each well. Basically, changing media everyday.
I am really confused as with both the case different results I am getting where using method 1 more viability is observed and with method 2 relatively very less viability of cells observed.
Kindly help me with what is the ideal way of preforming the MTT assay ?
There is no ideal way to perform the MTT assay. It measures cell death and there is a standard procedure (linked below). I am not sure I understand your 2 scenarios correctly. Regardless, I do not think the volumes or the frequency of media changes are causing the difference in viability. Your contradictory results could be coming from the different levels of cell death you have (depending on how you treat your cells with nanoparticles and how long the cells are exposed to NP before MTT measurement). Which scenario to use depends on your experimental objective and setup.
This is what I would recommend. Plate your cells in complete medium and leave them overnight to recover. Treat them with NPs (complete or incomplete medium depending on what you think is appropriate) the next day. If you need to, remove half the medium in the well first. Leave them for whatever it is that you expect to happen (1-24 hours depending on how long it takes). Proceed with MTT assay. You could do this in 2 ways.
1. Remove all media and rinse with PBS. This should remove nanoparticles/dead and floating cells. Add fresh media containing MTT.
2. Remove some media and replace with media containing MTT. The results may be confounded by NP/cells floating around in the culture medium.
If you are still unsure of your results, use an alternate cell death assay like Neutral red, PI/Annexin, G6PD/LDH release assays to verify your observations.
Julie Joseph thank you so much mam for the brief explanation. I'll definitely try to revise my protocol as you have suggested.
Happy to help. Just realized I forgot to add the link. Here it is.
https://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx
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