After a certain incubation period at high temperature, I am getting higher MTT readings as compared to smaller incubation periods at the same temperature. Cell viability should ideally decrease with time, MTT assay is a direct measurement of cellular metabolism, and works by using reducing equivalents such as the co-enzyme NADH to convert MTT into a colored formazan product. My question is: could the cells be producing some strange things after that period of time, that gives me higher absorbance. I take reading at 575 nm and 630 nm for background.
Or maybe formazan crystals can form outside the cells as well.....if plasma membrane get disrupted or something, but i don't think so because at even more incubation periods, readings are lower as expected..so something is happening only at that point of time. Should i use a different assay?
Dear Bhaskar, I am not an expert... but I performed the MTT assay a couple of times. Just a brief comment, there is an assay similar to MTT, but with a soluble compound.
Some nice information is in the next papers... "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture." and this one in which they use HeLa cells "MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets."
On the other hand, MTT crystals may be affecting the integrity of active cells because of crystal formation, possibly yielding lower readings than expected.
Here there is a nice picture indicating the size that crystals have under some conditions, see Fig. 4: "Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products of Vibrio splendidus", also I am attaching a picture from my experiments...
I'm a little unclear on the exact details of your experiment, but yes, the MTT assay can be affected by formazan crystals forming outside the cells - this is a problem one researcher in our department has come across recently. We've also had some rather strange results with primary cell lines with MTT in the past.
For HeLa cells (or most cancer cell lines) for example, you should have to incubate with MTT for no more than an hour at 37°C; if your experiment requires a higher temperature for some reason, this time would be shorter. If you leave it too long, you might affect your results.
Although the MTT assay is very useful it does have its limitations, and you may find a different assay works better for you. There are many alternatives, and selecting a better one will depend on exactly what you are following in your experiment. For example, if you want to read at multiple time points in the same assay in the presence of the dye, alamar blue would probably be a better choice. Good luck!
Dear Bhaskar, as an alternative way for measuring cell viability, you may try the MTS assay. In my experiment with MV4-11 (acute myeloid leukemia cell line) I used CellTiter96AQueous One Solution Cell Proliferation Assay (Promega) for this purpose. This kit contains MTS and an electron coupling reagent (phenazine ethosulfate, PES). The latter allows MTS tetrazolium compound to be bioreduced by cells into a colored formazan product that is SOLUBLE in cell culture medium. We added 20 microliter MTS solution to each well and incubated at 37C for 2h. Recording the absorbance at 490nm. It's said that incubation time may be 1-4h. In terms of experimental conditions one can see what time would be better to get an appropriate data. As for the MTT assay, I have never applied it before. However, recent report speculated that formazan crystals can exacerbate cell injury by exocytosis (http://www.ncbi.nlm.nih.gov/pubmed/22401948). The other thing, culture conditions that alter the metabolism of the cells will affect the rate of MTT reduction into formazan. I hope my answer will help you somehow to solve your problem. Best luck!
Thank you everybody. @Mark: I am not using a prmary cell line. I heat shock the cells at a higher temp and then do the MTT assay @ 37 deg for 2-3 hours, becuase thats when i get the crystals. One hour is somewhat less. See the thing is everything is the same for all the plates and wells at all othe rincubation points. So why after that T time is it showing anomaly? say ihave four time points @ higher temp ( 6, 12, 18,25)...so why 18 is hsowing anomaly?? giving me so much higher readings..a sif it had not been given heat shock at all....formazan crystals are forming too much it means at that particular condition. .... i would try a different assay lik,e the one suggested by Yulia. For now though, i have different experiments to do at 6 minutes only..the others were just for trials and learning the MTT per se. Iwould get back if and when i do some other assay . we do not have much money for playing around with interesting stuff unfortunately.:D
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