In the plot (Calcein vs Etidium), generally, I can see the dead cells in the upper-left position (cells killed using heat 56°/20 min) and the live cells in the down-right position. However, when I treated my cells with different cytotoxic compounds, the populations move slightly into the different quadrants. Do you think that in the using of this staining, the most important criteria to identify live and dead cells, is the position of cells and not its position into the quadrants?
Both Populations, are invading the quadrant calcein + / + and Ethidium
I find that viability assays tend to follow a path around the plot based on how far along in apoptosis the cells are. For example, I've seen in many of my stable cells that express a fluorescent protein that cells undergoing apoptosis 'travel' from PI-/FP+ to PI+/FP+ to PI+/FP-.
It could be that your treatments induce apoptosis at a much slower rate than the heat-shock induction and so what you are seeing is your cell population following a similar 'death path'. You could try testing a set of time points to confirm if this is the case.
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