10 replies to this topic

[netron]

I use GFP reporter gene to access promoter activity in living cells and use flow cytometry to quantitate GFP positive cells after treatment. For the FACS analysis, do I need to fix my cells like what we do for PI staining? If fixation is necessary, what method of fixation is appropriate?

Thank you.

[yobou]

yes you need to fix the cells in formaldehyde (0.25%) to avoid leaking of GFP after triton premeabilization.

[pcrman]

We have done FACS analysis of GFP expressing cells without fixation. We just harvest the cells, spin down and resuspend in PBS, then run FACS on FL1. I am not quite sure whether fixation is necessary.

[WOW]

pcrman, on Mar 14 2009, 11:51 PM, said:

We have done FACS analysis of GFP expressing cells without fixation. We just harvest the cells, spin down and resuspend in PBS, then run FACS on FL1. I am not quite sure whether fixation is necessary.

If you simply analyze changes of percentages of GFP+ cells, you could do it without fixation like pcrman suggests. If you need to stain with another intracellular antibody, and triton was thereby used, you need to fix the cell. May it help you and good luck.

[little mouse]

no need to fix, unless you use a viral vector, then it's better to fix to avoid viral vector spreading.

[memo]

I only fixate my cells if I have so much samples that I could not measure within the hour.
I fixate the cells with 0.25% paraformaldehyd in PBS1x after incubation with PI stain.

[netron]

Thank you friends for all your tips and guidance. I think I will just ran FACS without fixation because I only want to know the percent of GFP positive cells.

[gradbiotech]

pcrman, on Mar 14 2009, 08:51 AM, said:

We have done FACS analysis of GFP expressing cells without fixation. We just harvest the cells, spin down and resuspend in PBS, then run FACS on FL1. I am not quite sure whether fixation is necessary.

Its is advisable to add 2% FBS 9in PBS/HBSS) for better/longer stability of cells.

[zodiac1505]

I would suggest that you don't fix the cells and use them directly. I have GFP positive mice and I do FACS regularly for GFP +ve cells. I have also tried fixing the cells to stain for transcription factors. But once I fixed the cells, I find that GFP expression is lost and the cells look like WT GFP -ve cells. So now I don't look at GFP if I am doing fixation.

[SciCell]

zodiac1505, on 01 July 2010 - 06:46 AM, said:

I would suggest that you don't fix the cells and use them directly. I have GFP positive mice and I do FACS regularly for GFP +ve cells. I have also tried fixing the cells to stain for transcription factors. But once I fixed the cells, I find that GFP expression is lost and the cells look like WT GFP -ve cells. So now I don't look at GFP if I am doing fixation.

Hello all,

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.

[little mouse]

Sorry for the late reply.
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.