6 replies to this topic

[acute]

dear all,
I stain my cells with DAPI to show the apoptosis process in several times. However I am in doubt of the what I should see as apoptotic cells. in the late hours the apoptotic bodies are obvious. But what are the differences between viable cells and the cells in the early stage of apoptosis????
could anybody explain me on the uploaded photo?
thanks in advance...

Attached Files

•  P3082545.JPG   1.98MB   52 downloads

[Curtis]

I've been doing DAPI staining for only a year now but I know it is hard to differentiate viable cells from cells in their early stages of apoptosis by DAPI method.

when cells die their nuclei size decreases and you must see a higher intensity of DAPI from dead cells. I use 1-10ug/ml DAPI for less than one minute and I can see brighter blue color being emitted from dead cells compared to live cells but this is very difficult for you to detect as long as you are very new to it. viable cells have very round and clear nuclei..whereas dead cells have smaller, condensed, cut or chopped nuclei. however it depends on the cell type and the method of apoptosis induction too.

from what I see, your cells might be in their early stages of apoptosis or might be not (but definitely not in their late apoptosis).

you don't have much cells in your sample. next time grow more cells so you can compare them better because usually not all cells die at the same time!!

[NemomeN]

You definitely have 1 that looks apoptotic.  Generally, the dapi stain begins to look bumpy, like warts on a toad, but in shades of blue rather than brown

I would not use DAPI as your main marker to gauge apoptosis, but rather as a rough guide.

Interestingly, the early phenotype of cell division looks similar to early apoptosis, so beware.

[WOW]

I use Hochest rather than DAPI for nuclear staining, which should be similar. According to my personal experience, none (or at most only one) of cells in your photo were in apoptosis. I found apoptotic cells (late phase) have a seperated nuclei composed by clusters of blue dots (apoptotic bodyes?) and the nuclei was smaller than that in normal condition.
By the way, I think apoptotic body based on nuclear staining was not good for quantitative anaylsis of cells apoptosis. Cleaved-caspase would be better. If you are interested in early phase apoptosis, you could try dye for mitochondrial membrane potential or Annexin-V-FITC. May it help you and good luck.

[acute]

Dear All,
thnaks for ur helps. I cannot find any apoptotic bodies in my samples while annexin V assay shows 93% of apoptosis!!!!!!
I tried annexin V staining but i was unable to take photos it was tooo dark...
once again could I ask u to say ur experiences from this photo?
tooo sorry to disturb u all, but I am really alone in this field

[acute]

sorry, I meant this photo!

Attached Files

•  P3162609.JPG   1.88MB   37 downloads

[NemomeN]

Your data seems to be correct.  DNA blebbing is a late to very late phase of apoptosis while phosphatidylserine flipping is an early stage marker so you Annexin V result and your lack of DAPI result is consistent.