Before starting the staining procedure, 1 × 105 cells were seeded in 6-well plates. At 24 h after seeding, each plate was treated at concentrations of 1000 µM for aspirin, 100 µM for clopidogrel, 100 µM for prasugrel, and 20 µM for ticagrelor for 0, 24, 48, and 72 h. Cell senescence was detected using a Senescence β-Galactosidase cell staining kit (Cat #9860, Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Briefly, the cell media was removed, washed with PBS, and then fixed with a fixative solution at room temperature for 10–15 minutes. Then, it was washed twice with PBS, β-Galactosidase staining solution prepared in advance was added, and it was incubated overnight at 37 °C without CO2. Blue color was observed under a microscope at × 200 magnification.
Senescence β galactosidase cell staining kit
Manufactured by Cell Signaling Technology
Sourced in United States
The Senescence β-galactosidase cell staining kit is a laboratory tool used to detect and quantify senescent cells. The kit utilizes the increased lysosomal β-galactosidase activity at suboptimal pH, a biomarker associated with cellular senescence, to stain and identify these cells.
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6 protocols using senescence β galactosidase cell staining kit
1
Cell Senescence Induction by Antiplatelets
2
Quantifying Senescence in MSC Cultures
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To evaluate the senescence of MSCs, β-galactosidase activity for BM-MSCs (passages 5, 8, and 11) and iPSC-MSCs (passages 5, 8, 11, 17, and 50) was detected using the senescence β-galactosidase cell staining kit (Cell Signaling Technology, Beverly, MA, USA). In brief, cells were seeded in six-well plates and fixed for 10–15 min after cells reached 60–70% confluence. After washing, the cells were incubated with β-galactosidase staining solution at 37 °C for 16 h. Pictures for the positive staining of the blue color under an inverted microscope (Leica, Wetzlar, Germany) were then taken. The mean number of β-galactosidase-positive cells was calculated from three fields chosen at random. There were three replicates per condition.
3
Senescence Induction and β-Galactosidase Assay
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TO and SH-TO cells treated with DOX (1 μg/ml) for 24 h and 96 h and left to recover until the seventh day were seeded at a concentration of 7,500 cells/well. A positive control consisted of cells treated with Bleocin™ (2.5 μg/ml) for 24 h and left to recover until the seventh day. In Bleocin™ experiments 50,000 cells/well were seeded. The Senescence β-Galactosidase Cell Staining Kit (Cell Signaling) was used according to the manufacturer’s instructions. The cells were examined under a light microscope (Olympus IX71) and those cells with blue staining were considered positive for β-galactosidase activity. We estimated the percentage of β-galactosidase staining by analysing 5 individual fields per well from three replicates.
4
Senescence Evaluation by SA-β-Galactosidase Assay
5
Senescence-β-Galactosidase Cell Staining
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Cell senescence was determined using a Senescence-β-Galactosidase Cell Staining Kit (Cell Signaling Technology) according to the manufacturer’s instructions. Briefly, we rinsed the plate with PBS and fixed cells in staining fixatives for 15 min at room temperature. Fixed cells were then washed twice with PBS and stained with fresh SA-β-gal staining solution overnight at 37°C. Cells that stained positive for SA-β-Gal were quantified and analyzed.
6
Senescence Assay for UCSCs and ADSCs
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To evaluate the cell senescence, a senescence β-galactosidase cell staining kit (Cell Signaling Technology, Danvers, MA, USA) was used to assay the UCSCs and infant ADSCs. Cells at passages 6-7 were seeded at a density of 1 × 10 5 cells per 6-cm dish and cultured for 5 days at 37°Cand 5% CO 2 . The senescent cells were detected by staining in blue following the manufacturer's instructions. The stained cells were observed under a microscope.
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