Hi Heda,
there are loads of publications on that. Are your cells autofluorescent or GFP marked? For my investigations I used a CLSM. The fluorescence of PI is stimulated by laser light (488nm), followed by emission of red light you can detect with the microscope. PI only enters cells with compromised membranes, means apoptotic or dead cells. Once in the cell, PI binds to DNA and RNA, thus enhancing the fluorescence. You will not see cells with intact membranes, less you use a (green or blue fluorescent) counterstain (for bacteria both stains are included in a staining set named BacLight, e.g.). I do not know if and how you can differentiate between early and late apoptosis, maybe ask a distributor of stainings (Life Technologies, Cell Signaling Technology,....).
We do not own a fluorescence microscope, but CLSM might be more easy to use for your investigations, anyway.
Best wishes,Annette
AO and PI are intercalating nucleic acid-specific fluorochromes which emit green and orange fluorescence, respectively, when they are bound to DNA. Of the two, only AO is a membrane-permeable, cationic dye that binds to nucleic acids of viable cells and at low concentrations causes a green fluorescence. PI is impermeable to intact membranes but readily penetrates the membranes of nonviable cells and binds to DNA or RNA, resulting in orange fluorescence. When AO and PI are used simultaneously, viable cells fluoresce green and nonviable cells fluoresce orange under the fluorescence microscopy. The criteria for identification are as follows: (i) viable cells appear to have green nucleus with intact structure;(ii) early apoptosis exhibits a bright-green nucleus showing condensation of chromatin in the nucleus; (iii) dense orange areas of chromatin condensation showing late apoptosis; and (iv) orange intact nucleus depicting secondary necrosis
Hi Neda,
If you have access to a FACS sorter, you can sort the different populations based on PI content and get a lot more data. You can also analyse the FACS graphs and get the percentages of all different populations; sub G1 (dead/dying), G1, S and G2 phases.
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