Biology-Talk

I’m guessing that a major reason for using slide-counting rather than FACS is lack of access to a FACS machine (or expensive departmental fees for FACS use).
If you have a FACS machine, go for it (skill in using a FACS is rarer than using a microscope, so will make you more “marketable” for future research jobs). Don’t let that stop you getting a few “pretty pictures” for any papers/posters though.

Cell adhesiveness could be a factor, I suppose… I just cannot see how off the top of my head (or without a more detailed description of what you’re actually doing).

For FACS/TUNEL, I’d still counterstain. Otherwise how will you calculate the % of apoptotic cells wrt the whole population.

Good luck.

In my opinion, people who do TUNEL by counting cells manually are people who don’t have access to a flow cytometer. The PI stain in this method is used in the fluorescence microscopy technique so that you can distinguish each cell easier, and you can look for nuclear degradation (multiple dots of DNA vs. one solid nucleus).

For flow you really don’t have to do the counterstain because your cells are in suspension and going past the laser one at a time, and the flow cytometer won’t be able to see the nuclear fragmentation anyway. Unless there’s some reason you need the DNA stain (looking at cell cycle of non-apoptitic cells?) you shouldn’t have to do it.

Another benefit to the flow cytometry method is that microscopy techniques are very user-dependant, 2 people can count the same slide and get different numbers depending on their classification technique to define an apoptitic cell (what’s background? what’s above background?) whereas with flow cytometry you loose a large part of that person to person bias.

Plus it’s way faster. Do you want to spend a few hours setting up and running flow, or a few days counting cells manually?

there can be actually good reasons why to use a glass slide. If you have tissue and want to know which type of cell is stained. You are less interested in the number of the cells, but in the staining pattern.
Not everybody is growing their cells. There are still people who work on whole organisms, e.g. developmental biology, pattern formation by cell death…
but if you want to count and you have cells in culture, I agree flow will be preferable.

i agree with the above answers as to whether to do FACS or slides. however, it may have to do with how bright the TUNEL stained cells are. i haven’t used it before, so i can’t speak to its intensity. if the stain is dim, you can expose your microscope camera for as long as you want and get a bright picture. however, the FACS will only look at each cells for less than a microsecond, so it’d better be bright. i’d do a dry run first.
as far as the need for counterstaining, it depends on if already dead (past apoptotic) cells will stain positive for TUNEL (and i don’t know that off the top of my head). if dead cells have lost the ability to stain positive in the assay, then you have a specific marker (unlike annexin V, which will bind to apoptotic and dead cells and is why you need to counterstain with PI). if it is like annexin V in its specificity, then you need to counterstain with PI. but make sure you do that first before you permeabilize the cells in the TUNEL assay — otherwise everything will be positive for PI.