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I was wondering if you can use cell viability test using live/dead for flow cytometry in lieu of MTT assay? Is this acceptable?
MTT -based analysis of apoptosis is based on the calibration curve data for light absorption in the restricted range of cell number (say from 1000 to 20000 cells). Using MTT you can get only ESTIMATED the percentage of dead cells when you compare your test data to the calibration curve. The protocol you can get from the manufacturer of your MTT product. And the MTT-based apoptosis data should indicate ( as said above)the state of metabolism on the level of mitochondria. Flow cytometry will give you nearly exact number of dead cells.Flow- is more precise method, and considered by many as most reliable method. What kind of cell death : you can define the type of apoptosis using specific flow markers (propidium iodine, annexin V, TMRM and others - see attached paper). Another advantage - you can separate apoptosis from necrosis and count how many cells are necrotic or apoptotic, or both. You can check other processes just in one samples at the one run. so - flow will give you more information and it will be reliable and statistically significant info
Yes, but it gives you slightly different information. MTT evaluates mitochondrial activity of cells (which correlates usually with the number of live cells). For flowcytometry there are multiple options of viability/cell count detection , most simple probably stainig by propidium iodide, which is a fluorescent analogy to trypan blue exclusion (live cells dont stain, dead ones incorporate the stain in the nucleus).
MTT -based analysis of apoptosis is based on the calibration curve data for light absorption in the restricted range of cell number (say from 1000 to 20000 cells). Using MTT you can get only ESTIMATED the percentage of dead cells when you compare your test data to the calibration curve. The protocol you can get from the manufacturer of your MTT product. And the MTT-based apoptosis data should indicate ( as said above)the state of metabolism on the level of mitochondria. Flow cytometry will give you nearly exact number of dead cells.Flow- is more precise method, and considered by many as most reliable method. What kind of cell death : you can define the type of apoptosis using specific flow markers (propidium iodine, annexin V, TMRM and others - see attached paper). Another advantage - you can separate apoptosis from necrosis and count how many cells are necrotic or apoptotic, or both. You can check other processes just in one samples at the one run. so - flow will give you more information and it will be reliable and statistically significant info
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