Dec 25, 2025
Senescence β-galactosidase staining assay in cultured cells
- Xin Xu1,
- Yujuan Wang1
- 1Princess Margaret Cancer Centre
Protocol Citation: Xin Xu, Yujuan Wang 2025. Senescence β-galactosidase staining assay in cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp188jgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 21, 2025
Last Modified: December 25, 2025
Protocol Integer ID: 235613
Keywords: cellular senescence, beta-galactosidase, senescence assay, SA-beta-gal, cell aging, cancer biology, microscopy, cellular senescence by light microscopy, galactosidase, assay in cultured cell, cultured cell, senescence, staining assay, cell, light microscopy
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes senescence-associated β-galactosidase staining in cultured cells using a commercial staining kit to assess cellular senescence by light microscopy.
Materials
- Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, 9860)
Troubleshooting
Safety warnings
- Staining time may vary depending on cell type and senescence level.
- Do not use a CO2 incubator during the staining step, as CO2 can alter pH.
- Include untreated and positive control samples for accurate interpretation.
Before start
- Perform all steps at room temperature unless otherwise indicated.
- Prepare thestaining solution fresh before use.
- Use a dry incubator without CO2 for the staining reaction.
- Handle fixative and X-gal substrate according to institutional safety guidelines.
Cell preparation and positive control
Culture cells under experimental conditions until ready for senescence analysis.
(Optional positive control) Treat cells with 1 µM doxorubicin for 6 h prior to fixation to induce senescence-associated β-galactosidase activity.
Fixation
18m
Wash cells once with PBS.
1m
Fix cells with 1x Fixative Solution (provided in the Senescence β-Galactosidase Staining Kit, Cell Signaling Technology, 9860) for 10–15 min at room temperature.
15m
Rinse cells twice with PBS.
2m
β-galactosidase staining
Prepare fresh β-galactosidase staining solution (pH 6.0) containing X-gal substrate according to the manufacturer’s instructions.
Add staining solution to completely cover cells.
Incubate cells at 37°C in a dry incubator without CO2 for 72 h.
Imaging and analysis
Examine cells under a light microscope.
Identify senescent cells by the presence of blue staining.
Expected result
Senescent cells exhibit clear blue β-galactosidase staining, whereas non-senescent cells remain unstained.