Protocol Online logo
Top : Forum Archives: : Flow cytometry

Cell cycle analysis (FACS) analysis and GFP detection - (May/02/2006 )

Pages: 1 2 Next

Hi,


I would llike to perform following experiement


1.) Infect MEF cells with a GFp containing construct to distingusih infected vs noninfected and so target gene expressing cells.

2.) Perform cell cycle analysis with the GFP+ cells vs. non gfp controlls

Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Any ideas how to design the experiement. I know there are other dyes , like Hoechst 33342 that aloow for intravital staining, so the GFP should be functional as well (right?), any help is appreciated.

Oliver

-oschmah-

QUOTE (oschmah @ May 3 2006, 04:46 AM)
Hi,




Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Oliver

i have done the similar expriment as you said,but i wonder about waht you said above that "the fixation process kills the GFP (70% ethanol) ",can you explain it more detail

-bobxiang4-

I don't know if it's possible for this kind of experiment, but maybe fixation with PFA would help?

-vairus-

QUOTE (bobxiang4 @ May 2 2006, 10:02 PM)
QUOTE (oschmah @ May 3 2006, 04:46 AM)

Hi,




Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Oliver

i have done the similar expriment as you said,but i wonder about waht you said above that "the fixation process kills the GFP (70% ethanol) ",can you explain it more detail



GFP losses its green fluorecence when fixed, to my knowegde and experience it will not work in dead cells after fixing it (at least with ethanol)

-oschmah-

I use PFA fixation (as most people at our lab do) and have had my cells fixed for several days before doing FACS without any problem. Don't know about ethanol fixation though.

-vairus-

QUOTE (oschmah @ May 4 2006, 06:43 AM)
QUOTE (bobxiang4 @ May 2 2006, 10:02 PM)

QUOTE (oschmah @ May 3 2006, 04:46 AM)

Hi,




Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Oliver

i have done the similar expriment as you said,but i wonder about waht you said above that "the fixation process kills the GFP (70% ethanol) ",can you explain it more detail



GFP losses its green fluorecence when fixed, to my knowegde and experience it will not work in dead cells after fixing it (at least with ethanol)

in my experience,GFP green flurescence can sustain several days even though after fixing it,sometimes,we can detect green flurescene after a week.

-bobxiang4-

QUOTE (bobxiang4 @ May 4 2006, 11:17 AM)
QUOTE (oschmah @ May 4 2006, 06:43 AM)

QUOTE (bobxiang4 @ May 2 2006, 10:02 PM)

QUOTE (oschmah @ May 3 2006, 04:46 AM)

Hi,




Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Oliver

i have done the similar expriment as you said,but i wonder about waht you said above that "the fixation process kills the GFP (70% ethanol) ",can you explain it more detail



GFP losses its green fluorecence when fixed, to my knowegde and experience it will not work in dead cells after fixing it (at least with ethanol)

in my experience,GFP green flurescence can sustain several days even though after fixing it,sometimes,we can detect green flurescene after a week.


I have been trying to do the same thing. here is what I have come up with.

1) it is possible to do FACS with cells expressing GFP. I used 2% PFA fixation followed by membrane permeabilization with 70% EtOH. Then stain with PI and run FACS. (Curr Protocols in Cytometry, Unit 7.16.2).

2) I am trying an alternative method by transfecting a GFP tagged nuclear protein. This will allow for visualizing transfected cells and allow me to use simple fixation and still be able to gate for GFP+ cells.

-dval7780-

This may sound dumb, but I'll try anyway: How about sorting the GFP positive cells first, and then test cell cycle with PI? This way, in the fixation proccess you don't have to worry about the flourescence of the GFP, simply because you KNOW now that all the cells you have are transfected cells.
If this is stupid and I misunderstood something, I apologize

-liflaf1-

hi,

i suggest you to use another color for the detection of infected cells (YFP?), then you could do a CFSE co-staining wink.gif


Sebastien

-tryptofan-

QUOTE (dval7780 @ May 4 2006, 10:02 AM)
QUOTE (bobxiang4 @ May 4 2006, 11:17 AM)

QUOTE (oschmah @ May 4 2006, 06:43 AM)

QUOTE (bobxiang4 @ May 2 2006, 10:02 PM)

QUOTE (oschmah @ May 3 2006, 04:46 AM)

Hi,




Of course I know how to do "simple" cell cycle analysis by PI staining, but the fixation process kills the
GFP (70% ethanol)

Oliver

i have done the similar expriment as you said,but i wonder about waht you said above that "the fixation process kills the GFP (70% ethanol) ",can you explain it more detail



GFP losses its green fluorecence when fixed, to my knowegde and experience it will not work in dead cells after fixing it (at least with ethanol)

in my experience,GFP green flurescence can sustain several days even though after fixing it,sometimes,we can detect green flurescene after a week.


I have been trying to do the same thing. here is what I have come up with.

1) it is possible to do FACS with cells expressing GFP. I used 2% PFA fixation followed by membrane permeabilization with 70% EtOH. Then stain with PI and run FACS. (Curr Protocols in Cytometry, Unit 7.16.2).

2) I am trying an alternative method by transfecting a GFP tagged nuclear protein. This will allow for visualizing transfected cells and allow me to use simple fixation and still be able to gate for GFP+ cells.



Could you add some more detail?


what cells did you use?
how long PFA 2% treatment?
how long Ethanol fixing??

what is your staining buffer PI composed of?

thanks
Oliver

-oschmah-

Pages: 1 2 Next