Hi,
I am planning to do MTT assay with H69 lung cancer cell line in 96-well plates. These cells grow as floating aggregates. Is there any method to do the assay without removing any liquid from the plates? Or is it necessary to remove the liquid, wash and then solubilize the formazan crystals?
Thanks in advance,
Asrar
Dear Asrar,
in the original publication of the MTT assay by Mosmann ( Article Rapid Colorimetric Assay For Cellular Growth And Survival - ...
), a mixture of 0.04 N HCI in isopropanol was simply added to the wells and mixed to dissolve the dark blue crystals. However, many researchers (including me) tend to aspirate off the supernatant and replace it with DMSO nowadays, as this is a more efficient way for the extraction. In your case, I would recommend to switch to a tetrazolium salt, that generates water-soluble formazan crystals, e.g. WST-1 or XTT. For a closer look at a comparison of different reagents and potential pitfalls, also read the following discussion: https://www.researchgate.net/post/Best_reagent_for_cell_viability_asay-MTT_XTT_MTS_WST-1Good luck - CL
Dear Asrar,
in the original publication of the MTT assay by Mosmann ( Article Rapid Colorimetric Assay For Cellular Growth And Survival - ...
), a mixture of 0.04 N HCI in isopropanol was simply added to the wells and mixed to dissolve the dark blue crystals. However, many researchers (including me) tend to aspirate off the supernatant and replace it with DMSO nowadays, as this is a more efficient way for the extraction. In your case, I would recommend to switch to a tetrazolium salt, that generates water-soluble formazan crystals, e.g. WST-1 or XTT. For a closer look at a comparison of different reagents and potential pitfalls, also read the following discussion: https://www.researchgate.net/post/Best_reagent_for_cell_viability_asay-MTT_XTT_MTS_WST-1Good luck - CL
I'm agree with Cartsen that you can replace the mtt with xtt or ws-1 assays ( https://www.intechopen.com/books/genotoxicity-a-predictable-risk-to-our-actual-world/in-vitro-cytotoxicity-and-cell-viability-assays-principles-advantages-and-disadvantages) that are based on soluble colorimetric probes relased into the media? you can just centrifuge the cells, transfer the surnatant in a reading plate and read it
Manuele
Carsten Lange Thank you very much for the informative answer. It would be really helpful.
Manuele Martinelli Thank you very much for your input and the link.
- Badges
- Science topic