Viability

Cell viability can easily be determined in flow cytometry by adding one of DNA binding dyes at relatively low concentration to a population of cells. A common appproach is to use propidium iodide (PI) and 5 mg/ml; but there is a wide selection of dyes that can be used, which include 7-AAD, DAPI, DRAQ7 (Biostatus), SYTOX ADDVanced and To-Pro-3 all of these have to be used with caution as live cells will eventually take up these dyes if left to long, see figure and protocols.

Determination of cell viability after fixing

The determination of cell viability when cells are infected with a pathogenic organism can be problematic given the requirement to fix the sample before flow cytometric analysis. However using the new Invitrogen reagent Violet Fluor-Reactive Dye allows the investigator to determine cell viabililty by flow cytometry by pre-incubation of cells with dye before fixing, see figure and protocol.

Early and late stages of cell death

A cell becomes necrotic when the plasma membrane integrity is compromised sufficiently to let viability dyes such as propidium iodide, DAPI or DRAQ7 into the cell. Late apoptosis or necrosis can be identified by SubG1 DNA analysis. Early necrosis can now be identified by the use of plasma membrane probe, Bis-oxonol. Dead or necrotic cells display a differential signal of Bis-oxonol. DNA analysis of high and low intensity Bis-oxonol events show that low intensity events have a greater level of DNA in the SubG1 area compared to the high intensity events, see figure. The side scatter of high and low intensity bis-oxonol events are also larger when the intensity of bis-oxonol is high and vice a versa. Thus dead cells can now be identified as early or late necrosis by a combination of a viability dye, Bis-oxonol,The side scatter of high and low intensity bis-oxonol events are also larger when the intensity of bis-oxonol is high and vice a versa. Thus dead cells can now be identified as early or late necrosis by a combination of a viability dye, Bis-oxonol, quantification of DNA and side scatter, see figure.