Cells were grown in 6-well plates, fixed with 0.5% glutaraldehyde (w/v) (Sigma) in PBS for 10-15 min, washed with 1mM MgCl2/PBS (pH 6.0) and then incubated with X-Gal staining solution (1 mg/ml X-Gal, Thermo Scientific, 5 mM K3[Fe(CN)6] and 5 mM K4[Fe(CN)6] for 8 hr at 37°C. Brightfield images of cells were taken using the DP20 digital camera attached to the Olympus CKX41 inverted light microscope. The percentage of SA-β-Gal positive cells was estimated by counting at least 100 cells per replicate sample facilitated by the “point picker” tool of ImageJ software (NIH) 44 (link).
Protocol detail
Quantifying Senescence-Associated β-Galactosidase
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Camera
Glutaraldehyde
Light microscope
Mgcl2
Replicate
X gal
Corresponding Organization :
Other organizations : MRC London Institute of Medical Sciences, Institut Pasteur, University Medical Center Groningen, University of Groningen, Biomedical Research Networking Center on Neurodegenerative Diseases, Pompeu Fabra University, Imperial College London, Genome Institute of Singapore, Inserm
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Variable analysis
independent variables
- Glutaraldehyde concentration (0.5% w/v)
- Percentage of SA-β-Gal positive cells
- Growth of cells in 6-well plates
- Fixing of cells with glutaraldehyde for 10-15 minutes
- Washing with 1mM MgCl2/PBS (pH 6.0)
- Incubation with X-Gal staining solution (1 mg/ml X-Gal, 5 mM K3[Fe(CN)6] and 5 mM K4[Fe(CN)6]) for 8 hours at 37°C
- Imaging of cells using Olympus CKX41 inverted light microscope with DP20 digital camera
- Counting of at least 100 cells per replicate sample using ImageJ software
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