I was wondering if anyone has used the MTT assay to determine viability in tissue slices. I couldn’t find much published research on this application.
I previously applied the assay to murine brain slices, following the protocol from: DOI: 10.1016/j.brainresbull.2012.02.005 PMID: 22387609
Viability was assessed over 30 hours in artificial cerebrospinal fluid (aCSF), and I observed a consistent decline over time—which worked quite well.
Now, I'm applying the same method to 300 µm-thick murine kidney slices, kept in Krebs-Henseleit Buffer. However, the results are inconsistent: instead of a gradual decline, I observe a sharp spike in absorbance at one time point (between 33–50 hours), followed by a decrease.
Has anyone successfully used the MTT assay for kidney slices or other non-neural tissues? I'd really appreciate any insights on protocols, buffers, or troubleshooting tips.
Thank you!
Bhavika Mahajan , hello.
Generally, MTT can be used to measure the overall metabolic activity of slices with a thickness of 150–400 µm. The abnormal increase in absorbance of your mouse kidney slices may be related to chemical reactions of culture medium components, inflammatory changes in tissues, decomposition, or stimulation of certain cell subpopulations. It is recommended to use LDH release, live-dead staining, or histological methods as controls for troubleshooting.
There are also controversies and uncertainties about the principle of MTT, such as which other organelles, enzymes, and molecules are involved in MTT reduction, the source of extracellular formazan crystals, and the cytotoxic effect of the MTT reagent itself.
The article PMID: 34884632 mentions that the increase in OD value may be the result of increased cell density rather than an increase in the number of cells itself. In addition to the increase in the number of cells, which will increase the total amount of formazan, higher cell density may also change the level of formazan produced in each cell by changing the nature of intercellular signaling and thus changing the enzyme activity of the cells.
If you find the specific reason, please contact me further. I am very interested in this phenomenon, thank you!
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Dear Lucian Miles , Thank you very much for your response—I truly appreciate your input.
I’m particularly interested in your reference to tissue decomposition. Could you kindly elaborate on how decomposition might lead to increased formazan production and absorbance? My understanding was that as tissue decomposes, viable cell numbers decrease, which would reduce the reduction of MTT to formazan.
Regarding your point about MTT cytotoxicity, I had assumed this effect to be consistent across time points, and thus not responsible for a sudden increase in absorbance between 33–50 hours. Similarly, I believe cell density is not a significant variable here since I’m working with acute slices rather than cultured cells.
To provide some context: I’m storing murine kidney slices in Krebs-Henseleit buffer at 4°C without culturing. For comparison, I previously used aCSF with brain slices and observed a consistent decline in viability over time. During the MTT assay, slices are incubated at 37°C for 20 minutes with MTT.
I also performed live/dead staining using DAPI and propidium iodide, which showed a continuous decline in viability—confirming that viability is not increasing at any time point. This further supports my concern that MTT absorbance values may not be reflecting true viability under these conditions.
Any additional insights you could share would be extremely helpful. Thank you again for your time and guidance.
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