Senescence-associated β-galactosidase (SA-β-gal) staining was performed using the Senescence-Galactosidase Staining Kit (MBL International Corporation, Catalog #JM-K320-250) according to the manufacturer’s protocol. In brief, cells were washed with 2 ml PBS and fixed in 2 ml fixing solution for 10 min at RT. Next, the cells were washed once in PBS to remove the fixing solution and incubated in freshly prepared SA-β-gal at 37°C overnight without CO2. Afterward, SA-β-gal-positive cells (senescent cells) were identified as blue-stained cells under standard light microscopy (20X) (OPTIKA Microscopes, Ponteranica, BG, Italy) and quantified with the ImageJ analysis software1 .
Senescence galactosidase staining kit
Manufactured by MBL Life Science
Sourced in United States
The Senescence-Galactosidase Staining Kit is a laboratory tool used to detect and visualize senescent cells. The kit provides a staining solution that reacts with the enzyme beta-galactosidase, which is a well-established marker of cellular senescence. The staining process allows for the identification and quantification of senescent cells in various experimental and research settings.
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4 protocols using senescence galactosidase staining kit
1
Senescence-Associated Beta-Galactosidase Assay
2
Senescence-Associated β-Galactosidase Staining
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Senescent cells were identified by using the Senescence-Galactosidase Staining Kit (MBL International Corporation, Catalog #JM-K320-250) according to the manufacturer’s protocol. Briefly, cells were washed twice with 2 ml phosphate buffered saline (PBS) (1X) and resuspended in 2 ml of fixing solution for 15 min at room temperature. Then, the cells were washed twice with PBS (1X), the staining solution was added and allowed to incubate overnight at 37 °C without CO2. Afterward, senescence-associated beta-galactosidase-positive cells (senescent cells) were identified as blue-stained cells under light microscopy (20X) (OPTIKA Microscopes, Ponteranica, BG, Italy) and quantified with the ImageJ analysis software (https://rsb.info.nih.gov/ij/ ).
3
Senescence-Associated β-Galactosidase Assay
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Senescence–Galactosidase Staining Kit (MBL International Corporation; Woburn, MA, USA) was used to detect senescent cells. Cells were fixed and washed twice with PBS. After that, staining solution was added and cells were incubated overnight at 37 °C without CO2. Afterward, senescence-associated beta-galactosidase-positive cells (senescent cells) were identified as blue-stained cells using an inverted microscope (OPTIKA Microscopes, Ponteranica, Italy) in three–five random fields for each well and quantified with the ImageJ software.
4
Senescence-Associated β-Galactosidase Assay
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SA-β-gal staining was performed using the Senescence-Galactosidase Staining Kit (MBL International Corporation, Catalog #JM-K320-250) according to the manufacturer’s protocol. In brief, endothelial cells at different passages were plated in a 12-well plate (6 × 104/well) and analyzed when they reached confluency. The cells were washed with PBS and fixed in fixing solution for 12 min at room temperature. Next, they have washed once again in PBS to remove the fixing solution and incubated in freshly prepared SA-β-gal at 37 °C for 16 h without CO2. Afterward, SA-β- gal-positive cells (senescent cells) were identified as blue-stained cells under standard light microscopy. The number of positive cells with blue color was counted and normalized to the number of total cells in the same field. The percentage of SA-β-gal-positive cells was counted in 10 randomly selected microscopic fields (magnification 100x; 400–600 cells).
SA-β-gal activity was also measured by fluorescence microscopy. After the experiment, cells were incubated with C12FDG (fluorogenic substrate, 5-dodecanoyl-aminofluorescein di-β-D-galactopyranoside; 33 µM, Invitrogen) at 37 °C for 30 min. The images (between 7–10 for each condition) were analyzed using Image Pro-Plus software (Media Cybernetic).
SA-β-gal activity was also measured by fluorescence microscopy. After the experiment, cells were incubated with C12FDG (fluorogenic substrate, 5-dodecanoyl-aminofluorescein di-β-D-galactopyranoside; 33 µM, Invitrogen) at 37 °C for 30 min. The images (between 7–10 for each condition) were analyzed using Image Pro-Plus software (Media Cybernetic).
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