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

Posted on Monday, October 27, 2003

Cell Staining for Senescence-Associated -Galactosidase (SA--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 (see Hint #2).

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 37C (do not incubate in a CO2 incubator; see Hint #3).

Staining Solution 1 ml of 100 mM K3Fe(CN)6 (final concentration 5 mM)
40 l of 1 M MgCl2 (final concentration 2 mM)
*Add just before use
600 l 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! see Hint #1)
20 mg/ml X-Gal (5-bromo-4-chloro-3-indoly -D-galactopyranoside)
Store -20C
X-Gal is not stable in water and should be added to the Staining Solution just before use

100 mM Potassium Ferricyanide 100 mM Potassium Ferricyanide (K3Fe(CN)6)
CAUTION see Hint #1

100 mM Potassium Ferrocyanide 100 mM Potassium Ferrocyanide (K4Fe(CN)6)
CAUTION see Hint #1

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

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


1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. Cells can also be fixed in 3% (v/v) Formaldehyde in PBS; however the Fixative Solution preserves morphology better.

3. 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 -galactosidase activity.

Submitted by: manny440

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