Abstract
Infecting mammalian cells with a GFP construct to overexpress or knockdown target genes is one of the most commonly used methods to study and manipulate gene expression. To determine the target gene function on the cell cycle, analyzing the cell cycle (propidium iodide, PI staining) of GFP positive cells vs GFP negative cells is needed. Usually simple fixation of cells with 70% EtOH for PI staining tends to quench GFP signal; paraformaldehyde (PFA) fixation before ETOH fixation could help to sustain the GFP signal.
Keywords: Cell cycle, GFP, FACS, Propidium iodide
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This work was supported by the California Institute of Regenerative Medicine, Grant RL1-00100.
References
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I have used GFP antibody in Immunofluorescence staining. For FACS, I have never tried. If GFP+ % is fairly high, you don't need to add antibody. If GFP+ % is very low, adding antibody may not help.
PI is much more broader which fluoresces everywhere from yellow to far-red.You need to do compensation when go with GFP. You can use 7-AAD in place ofPI, which is more tighter.
Paraformaldehyde is godd to penetrate cells. The disadvantage is that paraformaldehyde Only partially destroys osmotic properties of membranes. Osmolarity must be carefully adjusted by glucose or sucrose.