Cell Staining for Senescence-Associated β-Galactosidase (SA-β-Gal

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Tony Rook
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Cell Staining for Senescence-Associated β-Galactosidase (SA-β-Gal

Please find the following protocol:

Cell Staining for Senescence-Associated β-Galactosidase (SA-β-Gal) Activity


1. Carefully remove the growth media from the cell culture.

2. Add an equal volume of PBS and gently mix the PBS solution around to wash out the cell culture media.

3. Repeat the PBS wash of the cells two more times.

4. Discard the PBS wash solution.

5. Add Fixative Solution to the cells and incubate at room temperature for 3 to 5 min. Cells can also be fixed in 3% (v/v) Formaldehyde in PBS; however the Fixative Solution preserves morphology better.

6. Wash cells as before in PBS two times.

7. Add between 1 to 2 ml of Staining Solution per 35 mm culture dish or cover slide.

8. Incubate for at least 2 hr at 37°C (do not incubate in a CO2 incubator). A blue color is visible within 2 hr; however, staining is maximum at between 12 to 16 hr. Blue stain is indicative of senescence-associated β-galactosidase activity.


Staining Solution
1 ml of 100 mM K3Fe(CN)6 (final concentration 5 mM)
40 μl of 1 M MgCl2 (final concentration 2 mM)
*Add just before use
600 μl of 5 M NaCl (final concentration 150 mM)
Prepare Staining Solution the day of experiment
4 ml of Citric Acid/Phosphate Buffer
(final concentration 30 mM)
1 ml of 100 mM K4Fe(CN)6 (final concentration 5 mM)
1 ml of X-Gal Solution* (final concentration 1 mg/ml)

Cell Fixative
in PBS
2% (v/v) Formaldehyde
0.2% (w/v) Glutaraldehyde

1 M MgCl2

5 M NaCl

X-Gal Solution
in DMF (CAUTION! Biohazard!)
20 mg/ml X-Gal
(5-bromo-4-chloro-3-indoly Β-D-galactopyranoside)
Store -20°C
X-Gal is not stable in water
Should be added to the Staining Solution just before use

100 mM Potassium Ferricyanide
100 mM Potassium Ferricyanide (K3Fe(CN)6)
CAUTION Biohazard

100 mM Potassium Ferrocyanide
100 mM Potassium Ferrocyanide (K4Fe(CN)6)
CAUTION Biohazard

Citric Acid/Phosphate Buffer
0.1 M Citric Acid
0.2 M Sodium Phosphate Dibasic (Na2HPO4)
Proper pH is VERY important
pH 6.0

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

Bioreagents and Chemicals

Potassium Ferricyanide (III)
Citric Acid
Potassium Ferrocyanide (II)
Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Magnesium Chloride

Reference and Links:

1. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et. al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U S A. 1995 Sep 26;92(20):9363-7.