Kindly confirm can we do MTT assay for quantification of apoptosis.
Is MTT Assay a valid assay to quantify apoptotic cells ?
What about LDH assay ?..how is it better for apoptosis quantification in cell culture experiments ?
Hello Asha Rao,
No, you cannot. Neither the MTT assay nor the LDH assay is related to apoptosis. The MTT assay measures the number of viable cells by relying on the intracellular reduction of MTT to formazan by mitochondrial dehydrogenases, while the LDH assay measures the enzyme activity of LDH in a sample to assess the extent of plasma membrane damage in a cell population.
Please note that DNA fragmentation is one of the hallmarks of apoptosis that is induced by apoptotic stimuli in cells. Flow cytometry is one of the most popular and versatile applications for studying apoptosis. It can be used to both detect and quantify the level of apoptosis in a population of cells at static points or in a time course.
If you are not able to use the flow cytometry, there are other methods you could use to measure apoptosis.
Most apoptotic process involves the activation of a cascade of caspases that mediate upstream signaling events ultimately leading to the activation of executioner caspases involved with destroying the cell. You could measure caspase-3/7 activity using consensus tetrapeptide substrates in lytic cell-based assay to generate a fluorescent or luminescent signal measured with a plate reader. You could try Western Blot analysis of your sample for pro- and active caspase as well as cleaved poly ADP ribose polymerase (PARP).
There are DNA fragmentation assays as well. You know that apoptosis and DNA cleavage go hand in hand. So, you could determine the extent of apoptosis by measuring the DNA fragments present in your sample. Apoptotic DNA ladder analysis, ELISA quantification and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay are some of the assays you could try.
Best.
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