Hello!
Have you ever observed that when measuring the viability of freshly thawed PBMC with trypan blue, the value is higher than with Annexin/PI staining or some other staining that takes into account early apoptotic cells? if yes, how large was the difference between the two methods? Which method is more reliable and will tell me the actual survival rate of the cells?
Thank you!
Hello Daniela Lascu,
1. The trypan blue method for cell viability may serve as a good indicator of cell membrane integrity, but it is not an optimal indicator of cell viability.
It does not differentiate between viable cells and cells that are losing cell functions (i.e., detection of necrotic cells only). When using trypan blue, under bright field microscope, cells that are very lightly stained with trypan blue can be hard to differentiate from unstained cells, and thus hard to identify. It can therefore give a higher viability count than what is expected. It would be difficult to compare different methods due to varying experimental conditions.
2. Cells have a relatively fixed amount of DNA/RNA. So, the DNA-interacting fluorophores like acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) will produce a uniform staining pattern for the quantification of cells, compared to the unspecified staining pattern when using bright-field based analysis.
AO is membrane-permeable and stains the total cell population (live and dead cells) whereas DAPI only stains the dead cell population. Therefore, fluorometric determination of cell viability using AO and DAPI provides superior accuracy to identify live and dead cells with reduced noise and variation. So, you try this method.
Best.
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