PI staining of adherent cells in plates for FACS - can this be performed in plates? (Nov/10/2006 )

Hi all!

Cell cycle analysis is quite new for me..so..
Can PI stain be done in plates for attached cells?
I´ve done this successfully in trypsinized cell suspension, however I wonder can this be done properly in plates also.
And how long have you done the ethanol fix in plates?
I was thinking to scrape the cells from plates after the staining.

Thanks in advance!

Joana

why would you want to do that?

stick with the protocol of trypsinising the cells, fixing them, and then staining them.

V

It would just be easier that way and more quicker , maybe I just have to test it.
I really don´t get why in our protocol the cells must be fixed with ethanol by vortexing.
In attached cells I could just fix them easier

scraping the cells would cause an increase in the sub G1 phase.
Fixing cells whilst they're still attached to the plastic/glass surface, and then scaping them off would increase the amount of membane damage. This could result in having false results (expecially when looking at PI or annexin V staining).
I'd rather spend 30 minutes extra trypsinizing and spinning the cells, than the time it would take the repeat an experiment because of inaccurate results.

V