SA-β-gal senescence staining solution (pH 6.0) recipe
Detects the β-galactosidase activity that rises in senescent cells as blue staining at pH 6.0 (senescence-associated β-gal).
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 0.2 M citric acid/sodium phosphate buffer pH 6.0 | 2 mL | 40 mM |
| Potassium ferrocyanide (K₄Fe(CN)₆·3H₂O) | 21.1 mg | 5 mM |
| Potassium ferricyanide (K₃Fe(CN)₆) | 16.5 mg | 5 mM |
| NaCl | 87.7 mg | 150 mM |
| 1 M MgCl₂ | 20 µL | 2 mM |
| X-gal 20 mg/mL (in DMF) | 500 µL | 1 mg/mL |
| Water (distilled) | to final volume |
Steps
- Dissolve everything except X-gal in water and confirm pH 6.0.
- Add X-gal just before use.
- Fix cells in 2% formaldehyde + 0.2% glutaraldehyde for 3–5 min and wash with PBS.
- Add staining solution and incubate at 37 °C without CO₂ for 12–16 h.
Good to know
- 0.2 M citric acid/phosphate buffer pH 6.0: 36.85 mL 0.1 M citric acid + 63.15 mL 0.2 M Na₂HPO₄.
- A CO₂ incubator acidifies the solution and stains non-senescent cells too.
⚠ Safety The X-gal solvent (DMF) is toxic — wear gloves.
FAQ
What if I incubate too long?
Beyond 24 h normal cells may stain faintly — compare with controls stained for the same time.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.