I have an issue. I need to measure cell viability of my HeLa (mammalian) cells that are in co-culture with bacterial cells (Prevotella, Lactobacillus). If used common MTT assay methods with a 96 well plate reader wouldn't the bacterial cells be able to generate the foramazan using non-mitochondrial methods?
Does anyone know of a dye that is specific to eukaryotic cells?
Also would flow cytometry be able to differentiate or sort the two cell types?
References to literature or webpages would be great. Thank you!
I do not know if the co-cultered bacteria is also present in the cytoplasm of the mammalian cells, I assume not because it would be impossible to differentiate otherwise.
1. Can you collect the cells by centrifugation and re-suspend them to measure viability afterwards by basic staining (tryphan blue or crystal violette)? As far as I know you can separate mammalian cells from bacteria by centrifugation.
2. Ki67-based viability assays are highly selective as they are antibody-based assays.
3. You can seperate cells from bacteria by flow cytometry. Since flow cytometry can measure size and granulation and use this data to cluster different cell types I believe same can be applied to your case as well. However I am unsure if you can measure bacteria with flow as they are relatively small.
You may find some alternatives here: http://www.abcam.com/primary-antibodies/the-cell-proliferation-guide
The bacteria have invaded into HeLa cell cytoplasm. If I separate and remove bacteria by centrifugation, then resuspend the cells in Media with Antibiotics would the bacteria still be able to interfere with assays?
Flow cytometry seems promising thanks.
Flow should work for this since if you want to omit the bacterial cells that have not invaded the cytoplasm from your analysis or if the University has a microscopy core with a High Content Imager, you should be able to generate this data quickly and easily by gating by size and will get additional info on your HeLa phenotypes, for instance you could monitor cell invasion into the cytoplasm, gate out the noninternalized bacterial cells and look at the relative cell counts with different parameters. High content will give you the most answers in the shortest time, with a fairly straight forward set of experiments. Good luck.
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