Self-brought instruments, reagents and consumables:
Microscope
without CO₂37 °C incubator
Pipette and tip, polypropylene tube (15 or 50 mL)
Various glassware for preparing reagents and buffer solutions
Deionized water, glycerol
Sealing film, 6-well plate
Reagent Preparation:
1 × PBS: Before use, dilute to 1 × PBS with deionized water and equilibrate to room temperature; Store at 4 °C.
1 × Fixation Buffer: Before use, dilute to 1 × Fixation Buffer with 1 × PBS and equilibrate to room temperature; Store at-20 °C.
Reagent A: ready-to-use; Before use, equilibrate to room temperature; Store at-20 °C.
Reagent B: ready-to-use; Before use, equilibrate to room temperature; Store at-20 °C.
Reagent C: ready-to-use; Before use, equilibrate to room temperature; Store at-20 °C.
X-Gal solution: ready-to-use; Before use, equilibrate to 37 °C; Store at-20 °C protected from light. It is very important to heat the X-gal solution at 37 °C for 1 h to avoid the formation of aggregates, which may interfere with the visualization of stained cells.
Staining Working Solution: Press 10 µ L Reagent A, 10 µ L Reagent B, 10 µ L Reagent C, 50 µ L X-gal solution And 920 µ L of 1 × PBS, then adjust to pH 6.0 with NaOH or HCl.
Note: When preparing the dyeing solution, please use containers made of polypropylene material or glass containers. A small amount of flocculation precipitation may occur and it will dissolve completely after shaking and mixing. Make sure it dissolves completely before use.

Experimental procedure:
Note: Staining Working Solution is toxic and corrosive to the human body. Please be careful when handling and pay attention to effective protection. Avoid contact with human body or direct inhalation.
1. For adherent cells:
1. Cultivate the cells directly on the coverslip of a 6-well plate and perform the required treatment.
2. Aspirate the cell culture medium and wash it twice with 1 × PBS.
3. Add 1 mL of 1 × Fixation Buffer to each well and fix at room temperature for 15 minutes. For other types of culture plates, the amounts of fixative and subsequent solutions are operated according to this ratio.
4. Aspirate the cell fixative and wash the cells 3 times with 1 × PBS.
5. Aspirate 1 × PBS and add 1 mL of Staining Working Solution to each well.
6. Without CO₂Incubate overnight in a 37 °C incubator until the cells stained blue.
Note: Seal the 6-well plate with a sealing film to prevent cells from drying out and select the appropriate staining time. Cells cannot be cultured in an incubator containing CO2 because cell senescence staining is pH-related。
7. Observe the cells under an optical microscope. The number of blue cells and total cells was counted, and the percentage of cells expressing beta-galactosidase (senescent cells) was calculated. After staining, if the counting cannot be observed in time, the Staining Working Solution can be removed, 2 mL of 1 × PBS can be added, and the cells can be stored at 4 ℃ for several days or covered with 70% glycerol solution for long-term storage at 4 ℃.

2. For suspension cells:
1. Centrifuge at 1,000 rpm for 5 min. Collect cells into 1.5 mL centrifuge tubes and wash twice with 1 × PBS.  
2. Add 1 mL of 1 × Fixation Buffer to each tube and fix at room temperature for 15 minutes. When fixed, it can be shaken slowly on the shaker to avoid the cells forming clumps.
3. At 1,000 rpm, centrifuge for 5 minutes, aspirate the cell fixative, and wash the cells 3 times with 1 × PBS.
4. Aspirate 1 × PBS and add 1 mL of Staining Working Solution to each tube.
5. Without CO₂Incubate overnight in a 37 °C incubator until the cells stained blue.
6. Take part of the stained cells, add them dropwise to a glass slide or a 6-well plate, and observe under an ordinary optical microscope. The number of blue cells and total cells was counted, and the percentage of cells expressing beta-galactosidase (senescent cells) was calculated. If the counting cannot be observed in time, the Staining Working Solution can be removed, 2 mL of 1 × PBS can be added, and the cells can be stored at 4 °C for several days or covered with 70% glycerol solution for long-term storage at 4 °C.

3. For tissue sections:
The paraffin sections were first dewaxed and hydrated according to conventional methods. Follow the following steps directly for frozen sections.
1. For the prepared tissue sections, add an appropriate volume of 1 × Fixation Buffer to fully cover the tissue, and fix it at room temperature for 15mim
2. Soak and wash the tissue with 1 × PBS for 3 times.
3. Aspirate 1 × PBS and add an appropriate amount of Staining Working Solution.
4. Without CO₂Incubate overnight in a 37 °C incubator until the cells stained blue.
5. Observe the tissue sections under an optical microscope. The number of blue cells and total cells was counted, and the percentage of cells expressing beta-galactosidase (senescent cells) was calculated. After staining, if it cannot be observed in time, cover the tissue sections with 70% glycerol solution and store them at 4 °C for a long time.

Cell is the basic unit of the structure and function of organisms, and it is also the basic unit of aging of organisms. Cell senescence is morphologically manifested as degenerative changes in cell structure, such as nuclear membrane depression, which eventually leads to nuclear membrane disintegration, chromatin structure changes, and an increase in the number of hyperdiploid and abnormal polyploid cells; The fragility of cell membrane increases, the selective permeability decreases, and the type, number and sensitivity of membrane receptors to ligands change; Lipofuscin accumulates in cells, and many organelles and intracellular structures undergo degenerative changes. The physiological manifestations of cell senescence are functional decline and low metabolism, such as cell cycle arrest, loss of cell replication ability, weakened responsiveness to mitogenic stimuli, and changes in responsiveness to pro-apoptotic factors; Intracellular enzyme active centers are oxidized, enzyme activity decreases, protein synthesis decreases, etc. Senescent cells are no longer able to replicate, but they remain metabolically active and are positive for senescence-related β-galactosidase activity, which is considered a biomarker of cellular senescence. The Cellular Senescence β-Galactosidase Staining Kit is a kit for staining and detection of senescent cells or tissues based on the up-regulation of the level of senescence-related β-galactosidase activity during senescence. Using X-Gal as the substrate, at pH 6.0, aging-specific β-galactosidase catalyzes the production of dark blue products, so that cells or tissues expressing β-galactosidase turning blue can be observed under light microscope. This kit is suitable for senescence detection of cultured cells and tissue sections, but stains only senescent cells, not presenescent cells, quiescent cells, immortal cells or tumor cells.
Product Components:

Own instruments, reagents, consumables:
microscope
incubator without CO₂37℃
pipet and tip, polypropylene tube (15 or 50 mL)
Various glass utensils for preparation of reagents and buffer solutions
deionized water, glycerol
sealing membrane, 6 orifice

reagent preparation:
1 & times; PBS: diluted to 1× with deionized water before use; PBS, equilibrated to room temperature; Store at 4 ° C.
1× Fixation Buffer: Before use, use 1× Dilute to 1× with PBS; Fixation Buffer, equilibrated to room temperature; Store at -20 ° C.
Reagent A: ready-to-use type; Before use, equilibrate to room temperature; Store at -20 ° C.
Reagent B: ready-to-use type; Before use, equilibrate to room temperature; Store at -20 ° C.
Reagent C: ready-to-use type; Before use, equilibrate to room temperature; Store at -20 ° C.
X-Gal solution: ready-to-use type; Before use, balance to 37 ° C; Store at -20 ° C away from light. It is very important to heat the X-gal solution at 37 ° C for 1h to avoid the formation of aggregates, which may interfere with the visualization of stained cells.
Staining Working Solution: press 10µ L Reagent A, 10 µ L Reagent B,10 µ L Reagent C ,50 µ L X-gal solution and 920 µ L 1× The reagent was mixed in the ratio of PBS and then adjusted to pH 6.0 with NaOH or HCl.
Note: When preparing the staining solution, use a container of polypropylene material or a glass container. A small amount of flocculated precipitate may occur, and it will dissolve completely after shaking and mixing. Make sure it is completely dissolved before use.

Experimental procedure:
Note: Staining Working Solution is toxic and corrosive to human body. Be careful when handling and pay attention to effective protection. Avoid contact with humans or direct inhalation.
I. For adherent cells:
1, the cells were directly cultured on a cover slip of a 6-well plate and treated as needed.
2, remove the cell culture medium and use 1× The cells were washed twice with PBS.
3, add 1 mL 1× to each well; The plates were fixed with Fixation Buffer for 15 min at room temperature. For other types of culture plates, the amount of fixation buffer and subsequent solution is operated according to this ratio.
4, remove the cell fixative and use 1× The cells were washed 3 times with PBS.
5, blot out 1× PBS, 1 mL Staining Working Solution was added to each well.
6, the cells were incubated overnight in a 37 ° C incubator in the absence of CO₂until they stained blue.
Note: Seal the 6-well plate with a sealing membrane to prevent drying of the cells, and choose the appropriate time for staining. It is not possible to grow cells in an incubator containing CO2 because the cell senescence stain is pH dependent.
7, the cells were placed under a light microscope for observation. The number of blue cells and total cells was counted, and the expression of &beta was calculated. -Percentage of cells expressing galactosidase (senescent cells). After Staining, if the Staining Working Solution cannot be observed and counted in time, the staining working solution can be removed and 2 mL of 1× added; PBS, 4 ° C can be stored for several days or cover cells with 70% glycerol solution at 4 ° C for long-term storage.

2. For suspended cells:
1, 1000 rpm for 5 min to collect cells into 1.5 mL centrifuge tube, and use 1× PBS  Wash 2 times.  
2, add 1 mL 1× to each tube; The tubes were fixed with Fixation Buffer for 15 min at room temperature. The cells can be fixed by slowly shaking them on a shaker to avoid clumping.
3, 1000 rpm, after centrifugation for 5 min, remove the cell fixative and use 1× The cells were washed 3 times with PBS.
4, blot out 1× PBS, 1 mL Staining Working Solution was added to each tube.
5, the cells were incubated overnight in a 37 ° C incubator in the absence of CO₂until they stained blue.
6, some of the stained cells were dropped onto a slide or inside a 6-well plate and observed under an ordinary light microscope. The number of blue cells and total cells was counted, and the expression of &beta was calculated. -Percentage of cells expressing galactosidase (senescent cells). If the Staining Working Solution cannot be observed and counted in time, the staining working solution can be removed and 2 mL 1× added; PBS, 4 ° C can be stored for several days or cover cells with 70% glycerol solution at 4 ° C for long-term storage.

III. For tissue sections:
For paraffin sections, deparaffinization and hydration were performed first according to conventional methods. For frozen sections, follow the following steps directly.
1, for prepared tissue sections, add the appropriate volume of 1× Fixation Buffer, appropriate to fully cover the tissue, fixed at room temperature for 15mim
2, with 1× The tissues were soaked in PBS and washed 3 times.
3, absorb for 1× PBS, and the appropriate amount of Staining Working Solution was added.
4, the cells were incubated overnight in a 37 ° C incubator without CO₂until they stained blue.
5, the tissue sections were placed under a light microscope for observation. The number of blue cells and total cells was counted, and the expression of &beta was calculated. -Percentage of cells expressing galactosidase (senescent cells). After staining, if it cannot be observed in time, cover the tissue sections with 70% glycerol solution and store them at 4 ° C for a long time.

Stored at-20 ℃, shelf life is 12 months.