FACS analysis of GFP expressing cells - Fix or not
Posted 13 March 2009 - 08:00 PM
Posted 13 March 2009 - 10:51 PM
Posted 14 March 2009 - 07:51 AM
Posted 16 March 2009 - 02:56 PM
Posted 17 March 2009 - 02:29 AM
Posted 17 March 2009 - 06:35 AM
I fixate the cells with 0.25% paraformaldehyd in PBS1x after incubation with PI stain.
Posted 17 March 2009 - 09:36 PM
Posted 02 March 2010 - 07:07 PM
Its is advisable to add 2% FBS 9in PBS/HBSS) for better/longer stability of cells.
Posted 01 July 2010 - 06:46 AM
Posted 21 September 2010 - 03:51 PM
I do the FACS for GFP without fixing but what if I harvest the cells today and run the FACS the next day after storing the cells at 4 degrees? Do I need to fix the cells?
Will there be any deterioration the the GFP expression? I Actually did this because of the FACS machine mal-funtion and I could not acquire my samples the same day as I harvested so I let them sit in staining buffer (0.1% BSA in PBS) overnight at 4 degrees and acquired the data the next day. Everything looked fine except that I expected higher data. What is the effect of fixing or not fixing the GFP expressing cells?
Thanks for your replies.
Posted 10 November 2010 - 07:01 AM
If you can go to the FACS immediately, you don't need to fix, otherwise fix with 0.5% PFA. It works fine for me.
If you have to go the day after, it's better to fix the cells.