Assay for Senescence-Associated β-Galactosidase

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Tony Rook
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Assay for Senescence-Associated β-Galactosidase

Please find the following protocol:

Assay for Senescence-Associated β-Galactosidase


This protocol describes a histological procedure for identifying replicative senescence in fibroblasts and keratinocytes that are stably expressing β-galactosidase. This assay takes advantage of the fact that a cellular β-galactosidase with a pH optimum of 6 is detectable as these cell types undergo senescence in culture.


1. Wash the cultured cells twice with PBS.

2. Fix the cells 3 to 5 min with 2% Formaldehyde/0.2% Glutaraldehyde at room temperature. You can also fix the cells in 3% Formaldehyde but the fixative given preserves morphology somewhat better.

3. Wash the cells twice with PBS.

4. Add 1 to 2 ml of Senescence-Associated β-Galactosidase Staining Solution per 35 mm dish.

5. Incubate at 37°C (NOT in a CO2 incubator).

6. View the cells under the microscope when staining is detectable. The blue color will be observable in some cells within 2 hr but maximal staining is usually not seen until 12 to 16 hr. Cells are best viewed by bright field microscopy, but the stain is also detectable under phase contrast microscopy.


Citric Acid/Sodium Phosphate, pH 6.0
126 mM Sodium Phosphate, Dibasic (Na2HPO4)
Check that the pH is 6.0
The pH of this buffer is important
36.8 mM Citric Acid

Senescence-Associated β-Galactosidase Staining Solution
Prepare in a total volume of 100 ml just before use
20 ml of Citric Acid/Sodium Phosphate, pH 6.0
150 mM NaCl
2 mM MgCl2
5 mM Potassium Ferricyanide
5 mM Potassium Ferrocyanide
1 mg/ml X-Gal

20 mg/ml X-Gal
Store at -20°C
20 mg/ml 5-Bromo-4-chloro-3-indolyl beta D-galactoside
Prepare in Dimethyl Formamide - CAUTION Biohazard!

2% Formaldehyde/0.2% Glutaraldehyde
2% (v/v) Formaldehyde - CAUTION Biohazard!
Prepare in PBS
0.2% (v/v) Glutaraldehyde - CAUTION Biohazard!

4.3 mM Sodium Phosphate, Dibasic (Na2HPO4)
pH 7.2
2.7 mM KCl
1.8 mM Potassium Phosphate, Monobasic (KH2PO4)
137 mM NaCl

Bioreagents and Chemicals:

Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Magnesium Chloride
Potassium Ferrocyanide
Potassium Ferricyanide
Dimethyl Formamide (DMF)
5-Bromo-4-Chloro-3-Indolyl β D-Galactoside (X-Gal)
Citric Acid
Potassium Phosphate, Monobasic

Reference Link: