I have several patient-derived cell lines that are slow-growing and material is limited, so instead of flow cytometry I wanted to try measuring MMP using a fluorescent microplate reader on 96-well plate. I've already optimized concentrations and incubation times with JC-1 and CCCP using a control line and measuring with flow cytometry. Using these parameters, I tried it also with a microplate reader using a cell culture black 96-well plate, and it seemed to work pretty well. I've noticed that most of the literature uses flow cytometry or fluor microscopy to obtain red/green ratios. Does anyone know if there is a good reason why I shouldn't use a plate reader? Would a reviewer have a reason to find it unacceptable?
A plate reader takes a bulk measurement of all cells in a well, whereas flow cytometry and fluorescence microscopy both take measurements that can be discriminated down to the single cell level. If only a sub-population of your cells end up exhibiting mitochondrial depolarization, then your signal-to-noise ratio may be too low to identify the cells of interest when you measure a bulk population. If all of your cells are expected to respond, then a plate reader may be fine.
I use a stirred cuvette-based spectrofluorophotometer as well as a microscope to do JC-1 measurements and the results are virtually identical. It just depends on your specific experiment and expected results.
It would be worth doing the experiment a few times with a microscope in parallel to the plate reader in order to validate that your results are complementary.
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