I am doing an apoptosis assay using dual staining with PA and Hoechst.
Regarding the calculation percent of apoptotic cells I am little bit confused;
Is this procedure right?
I have 3 types of population
1. Hoechst stained (bright blue) cells : Apoptotic cells
2. Both PI and Hoechst stained cells : Late stage of apoptosis
3. PI stained cells only (Red cells) : Dead cells
Calculation of % of apoptotic cells = Hoechst stained (bright blue) cells + Both PI and Hoechst stained cells / total no of cells - only PI stained cells) x 100
I agree but i don't understand than why there is need of PI staining for dead cells.
One important basic to remember in this matter : Please dont be biased in brightness of color: Hoechst / DAPI is practically nothing but the nuclear stain. If apoptotic, you can find lobbed (degraded nucleus) instead intact rounded one in case of non apoptotic control. Annexin V conjugated with flurochrome eg (Ann V-FITC/AnnV CY 3) can also be used to confirm which specifically binds with the exposed phophatidyle serin residue of plasma membrane which expose during early apoptosis .
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