Senescence was induced by spontaneous immortalization, etoposide treatment, or irradiation. Senescent cells were identified by a senescence-associated β-galactosidase kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. For senescence induced by spontaneous immortalization, MEFs during serial passages were harvested and fixed for SA-β-Gal staining. For etoposide-induced senescence, immortalized MEFs were treated with 10 μM etoposide for 24 h and cultured in normal medium for another 5 days. For IR experiments, MEFs were irradiated with 3 Gy and 6 Gy using an X-ray irradiator. The cells were harvested on day 6 for SA-β-Gal assays. Fresh tissues from 5-month-old WT, Trex1−/− and littermate controls were fixed in 4% Paraformaldehyde for 12 h and then transferred to 30% sucrose overnight. Tissues were then embedded in cryo-embedding medium (OCT) and cryosectioned at 8 μm for staining of SA-β-gal (pH 6.0) at 37°C for 16–24 h in SA-β-gal staining solution. Slides were imaged at 10× with upright microscope (ZEISS).
Senescence associated β galactosidase kit
Manufactured by Beyotime
Sourced in China
The Senescence-associated β-galactosidase kit is a laboratory tool used to detect the presence and activity of the senescence-associated β-galactosidase enzyme. This enzyme is a widely used biomarker for cellular senescence, a state of permanent cell cycle arrest. The kit provides reagents and protocols to perform the histochemical staining and quantification of senescent cells.
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5 protocols using senescence associated β galactosidase kit
1
Senescence Induction and SA-β-Gal Assay
2
Senescence Assessment of Passage 6 hWJ-MSCs
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Passage 6 hWJ-MSC from 2D and 3D cultures were seeded into six-well plates, with 1 × 105 cells/well. After 24 hours culturing, the cells were stained for senescence-associated β-galactosidase (SA-β-gal) to detect cell senescence, using the Senescence-Associated β-Galactosidase kit (Beyotime, Beijing, China) according to the instructions of the manufacturer. Senescent cells were observed using an optical microscope and counted in three random fields of vision (200 cells per field were used to calculate level of positivity) [44 (link)].
3
Senescence-Associated β-Galactosidase Assay
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SA-β-gal staining was performed using a senescence-associated β-galactosidase kit (Beyotime, C0602). A549 and H1299 cells seeded in 12-well plates were washed twice with PBS, fixed in fixative for 15 min, washed, and incubated overnight at 37 °C with the working solution of β-galactosidase plus X-Gal. The senescent cells were observed under an optical microscope (leika, DMi8) and counted from three random fields of vision.
4
Senescence-Associated β-Galactosidase Assay
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According to the manufacturer’s protocol of senescence-associated β-galactosidase kit (Beyotime, C0602), SLCs seeded in 12-well plates were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min. After washing, SLCs were incubated overnight with the working solution of β-galactosidase plus X-Gal at 37°C. The senescent cells were observed under an optical microscope (Leika, DMi8) and counted from three random fields of vision.
5
Senescence-Associated β-Galactosidase Staining
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Senescence-associated β-galactosidase kit (Beyotime, C0602) was used for SA-β-gal staining. LX2 cells were incubated with β-galactosidase and X-Gal overnight at 37 °C after fixation in fixative for 15 min. Optical microscope (Leica, DMi8) was applied for following observation of senescent cells.
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