Problem with X gal staining - (Aug/22/2006 )

Hi everybody,

I detected the senescent stage of MEF cells by using SA beta galactosidase assay referened from the article:

A biomarker that identifies senescent human cells in culture and in aging skin in vivo (Dimri et al.)

Briefly, cells were fixed for 5-10 mins in 2% formaldehyde + 0.2% glutaraldehyde, washed and incubated with fresh staining solution (1mg/ml of X-gal diluted in the staining buffer [40mM citric acid/sodium phosphate pH6.0, 5mM potassium ferrocyanide, 5mM potassium ferricyanide, 150mM NaCl, 2mM MgCl2] for 10-12h at 37oC without CO2. However, there is always the precipitation of X-gal (or something else, I'm not sure) in the well even though I warmed the staining solution before adding X-gal as some protocols recommended.

Is there anyone who has the experience with X-gal staining for animal cells? Please help me to solve the problem.

Thank you so much.

I noticed some crystals, rather than precipitation appeared during long incubation time (>4hrs). Fortunately, the number was not so many to raise a serious concern about it. I tried to avoid these crystals when taking a picture.