Apoptosis by PI staining on epithelial cells? - (Dec/07/2007 )

Hi all,

Hope you can help.
I'm trying to assess the proportions of cells that are apoptotic and in each cell cycle phase by PI staining and FACS. I have been using the following protocol:

Scrape cells from plates
Pellet and wash in PBS/2% FCS
Resuspend in 70% EtOH whilst vortexing
Store for at least 24 hours at -20C
Pellet from EtOH and wash in PBS/2% FBS
Incubate with 0.1mg/ml RNAse A, 50ug/ml PI, 0.05% Triton for 40 mins at 37C
Wash in PBS/FCS
Resuspend in FACS flow and analyse

The instrument settings I've used are FL2 at 470V and linear scale

I'm using a range of breast and colorectal cell lines but all show a similar profile.

The problem I'm getting is that even in the control, untreated cells, a large proportion appear dead (large amount of cells beyond Y-axis).
My questions are:
Is this peak actually sub-G1/apoptotic cells or is it just debris?
What changes to the protocol would people suggest to reduce what must be non-specific staining since I can't believe so many are actually dead!?

Many thanks for any help,

Giles

Hi Giles,

Do not scrape your cells from the plate as this disrupts the membrane and allows PI to enter all of your cells. The debris you are seeing is likely due to this harsh treatment. Use a gentler method to lift the cells from the plate i.e. Trypsin/EDTA or even EDTA alone.

Many thanks for your reply Penguin. Will give that a go this week

I also suggest a low number of rpm when you pellet your cells before the first wash with PBS/FCS 2%.
I mean, when I usually pass my cells I centrifuge them at 1000-1200 rpm for 10 min.
But when I pellet them for washing before EtOH fixation I centrifuge them at 800 rpm for 10 min.
Dead cells are "lighter" than living ones, so they will remain in the supernatan you'll discard.
Of course scraping is the main responsible for cell death, but this trick may help for a clearer result.