SA-β-gal activity is a widely used biomarker of cellular senescence [26 (link)] and was thus applied to identify KGN cell and mouse GC senescence. KGN cells and mGCs were seeded on 12-well plates at a density of 5∗105 cells/ml, followed by being washed with PBS and fixed and stained with X-gal solution (C0602, Beyotime Biotechnology) overnight at 37°C. Cells were imaged and photographed using an inverted microscope (Axio Observer A1, Zeiss, Germany).
X gal solution
Manufactured by Beyotime
Sourced in China
X-gal solution is a colorimetric substrate used in molecular biology and microbiology applications. It is commonly used for the detection and visualization of β-galactosidase activity in recombinant bacterial cells or tissues.
Automatically generated - may contain errors
4 protocols using x gal solution
1
Quantifying Cellular Senescence via SA-β-Gal
2
Senescence Induction in HT22 Cells
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Hippocampal neuronal HT22 cells exposed to sub-cytotoxic oxidative stress undergo stress-induced premature senescence, which may be identified using SA β-gal staining (18 ). HT22 cells were exposed to 100 mM tert Buty1 hydroperoxide (t-BHP) for 2 h at 37°C.
The SA β-gal staining kit (Beyotime Institute of Biotechnology) was used to detect senescent cells according to the manufacturer's protocol. Briefly, cells were seeded into 96-well culture plates (Corning Incorporated, Corning, NY, USA) at a density of 5×103 cells/well and cultured for 24 h. Then, the cells were washed twice with PBS and fixed in 1 ml 3% formaldehyde at room temperature for 15 min. Following fixation, cells were washed with PBS three times and stained with 1 ml freshly prepared cell staining working solution (containing 10 µl SA β-gal staining solution A, 10 µl SA β-gal staining solution B, 930 µl SA β-gal staining solution C and 50 µl X-Gal solution; obtained from Beyotime Institute of Biotechnology) at 37°C for 12 h in the dark. Finally, a light microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan) was used to identify the blue-stained cells.
The SA β-gal staining kit (Beyotime Institute of Biotechnology) was used to detect senescent cells according to the manufacturer's protocol. Briefly, cells were seeded into 96-well culture plates (Corning Incorporated, Corning, NY, USA) at a density of 5×103 cells/well and cultured for 24 h. Then, the cells were washed twice with PBS and fixed in 1 ml 3% formaldehyde at room temperature for 15 min. Following fixation, cells were washed with PBS three times and stained with 1 ml freshly prepared cell staining working solution (containing 10 µl SA β-gal staining solution A, 10 µl SA β-gal staining solution B, 930 µl SA β-gal staining solution C and 50 µl X-Gal solution; obtained from Beyotime Institute of Biotechnology) at 37°C for 12 h in the dark. Finally, a light microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan) was used to identify the blue-stained cells.
3
Rb Growth Suppression Assay
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For Rb growth suppression assays, Saos-2 cells were co-transfected with pCMV-Rb and/or PLVX-Pin1 (WT or mutant) by Lipofectamine 2000 (Invitrogen Inc.) and selected by puromycin resistance (0.5 μg/ml). Stable cells were grown in normal growth media for 10 days. Cells were then visualized under a light microscope and assessed for percentage of large, flat cell morphology. For senescence-associated β-Galactosidase (SA-β-Gal) staining, cells were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, washed and stained with X-Gal solution (Beyotime, Shanghai, China) for 16 h at 37 °C. Cells were then visualized under a light microscope and assessed for percentage of β-Gal-positive cells.
4
Senescence Detection in Neural Progenitor Cells
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SA-β-Gal staining is a commonly applied method to detect the cellular senescence. After treatment, NPCs were fixed by 0.2% paraformaldehyde for 15 min at ambient temperature. After being rinsed with phosphate buffer saline (PBS), NPCs were stained with X-gal solution (Beyotime, Wuhan, China) for 24 h at 37 °C. Senescent NPCs were observed with the use of an optical microscope (Olympus, Japan).
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