How to distinguish dead cells by DAPI staining?

[Dunia]

Hi, everyone!

I am doing IF staining on mouse splenocytes cultured for 1 to 7 days, as they start dying by apoptosis or some other manner by the end of culturing about 50% of cells on the slides are dead cells.

My question is how can I distinguish between dead and living cells by DAPI?

Cultured splenocytes are stained for IgMxbiot and strp-TR and counterstained with DAPI. Cells that I suspect for being dead ones sometimes have very bright red staining yet on DAPI filter they are visible but shadow like and the size of erythrocyte. Are they false positive for IgM because they are just cell debris?? When I stain normal splenocytes all their nuclei are large and evenly shaped and all as one show bright blue staining.

[richard03]

I would use TUNEL or Caspase-3 staining instead.

Richard

[Dunia]

Thanks for your reply Richard

As I understand, caspase-3 and TUNEL assay are used to detect apoptotic cells, but my question is more about nonspecific staining that a dead cell can give and if it is possible to distinguish such cell by DAPI.
Sorry I did not specify that I do double IF and use DAPI (third color) just for visualizing non-stained cells to make pictures representative.
On my slides with cultured splenocytes dead cells are abundant. I've heard they can give non-specific staining. And in fact there are cells or cell debris on slides that have bizarre staining pattern for my antigens though very bright and their nuclei are small and have very low DAPI staining comparing to the rest of  cells and to non cultured splenocytes. So I wonder if this shadow like DAPI staining is characterizing dead cells (being just some kind of background) and I can exclude them as false positive.  

PS When I do negative control without using primaries, such cells remain unstained.

[Pavel]

DAPI has about 4 times brighter fluorescent signal when bound to DNA than when it is free. To my knowledge they should not really be any background with DAPI if you do enough washes.
Sometimes you can see apoptotic cells with their characteristic fragmented nuclei.

[Dunia]

Thank you Pavel for your comments on DAPI staining
guess it was better to say autoflorescence instead of background, because those suspiciouse cells look like if I have not put DAPI at all but use DAPI filter to see them

Next time I'll try to search for apoptotic nuclei may be they will tell me something new (unfortunately I am not sure of how they should look like)


Dana

[Pavel]

Oh, it's quite simple, just as it sounds. Nuclei undergo fragmentation during apoptosis. That is instead of one nice and round nucleus you see several (commonly 3) small round fragments next to each other. When you see them, you won't miss 'em!