Cellular Senescence Detection Kit (SA-β-Gal Staining)

Catalog No. ABIN2344912

Product Details

Reactivity: Mammalian

Application: Detection (D)

Sample Type: Cell Samples

Components:

1. 100X Fixing Solution : One tube - 1.5 mL of 25% Glutaraldehyde
2. Staining Solution A : One tube - 1.5 mL of 500 mM Potassium Ferrocyanide
3. Staining Solution B : One tube - 1.5 mL of 500 mM Potassium Ferricyanide
4. Staining Solution C : Three tubes - 1.5 mL of 1 M Citrate-Na2HPO4 Buffer, pH 6.0, 50 mM MgCl2 in each tube
5. Staining Solution D : Two tubes - 2.0 mL of 5 M NaCl in each tube
6. X-gal Solution : Two tubes - 1.5 mL of 40 mg/mL X-gal in DMF in each tube 2

Material not included:

1. PBS
2. 37 °C Incubator
3. Light microscope
4. Senescent cells or tissue samples

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Stain cells for senescence-associated ß-galactosidase
• Assay performed in a standard 35 mm culture dish

Reagent Preparation:

• 1X Fixing Solution: Prepare a 1X Fixing Solution by diluting the provided 100X stock 1:100 in 1X PBS. Store the diluted solution at room temperature for up to six months.
• Cell Staining Working Solution: Prepare FRESH cell staining working solution based on the number of samples. The chart below is suggested for samples in 35 mm plate, and may be modified accordingly to suit other culture plate sizes. 1 dish 5 dishes 10 dishes Reagents (35 mm) (35 mm) (35 mm) Staining Solution A 20 μL 100 μL 200 μL Staining Solution B 20 μL 100 μL 200 μL Staining Solution C 80 μL 400 μL 800 μL Staining Solution D 60 μL 300 μL 600 μL X-Gal Solution 50 μL 250 μL 500 μL H2O 1.77 mL 8.85 mL 17.7 mL Total 2 mL 10 mL 20 mL

Assay Procedure:

1. Aspirate the medium from the senescent cells expressing SA-ß-Gal.
2. Wash the cells twice with 3 mL of 1X PBS and aspirate the final wash.
3. Add 2 mL of 1X Fixing Solution. Incubate at room temperature for 5 minutes.
4. Remove the fixing solution and wash the fixed cells three times with 3 mL of 1X PBS.
5. Aspirate the final wash, and completely cover cells by adding 2 mL of freshly prepared Cell Staining Working Solution.
6. Incubate the cells at 37 °C protected from light for 4 hr to overnight.
7. Remove the Cell Staining Working Solution, then wash the stained cells twice with 3 mL of 1X PBS and store cells in 1X PBS. For long-term storage, overlay the cells with 1X PBS containing 20 % Glycerol. Store at 4 °C. Note: Excess amount of salt crystals can be removed by briefly incubating the stained sample with DMSO. 3
8. Count the blue stained senescence cells using light microscope.

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: Store X-gal solution protected from light at -20°C. Store all other components at 4°C.

References

OHara, Splinter, Trussoni, Pisarello, Loarca, Splinter, Schutte, LaRusso: "ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A." in: The Journal of biological chemistry, Vol. 292, Issue 12, pp. 4833-4846, (2017) (PubMed).

Rao, Atay, Shukla, Hong, Upham, Ripley, Hong, Zhang, Reardon, Fetsch, Miettinen, Li, Peer, Sissung, Figg, De Rienzo, Bueno, Schrump: "Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 22, Issue 5, pp. 1197-210, (2016) (PubMed).

Kim, Ko, Lee, Park, Park, Kim, Sung, Koo, Yoo: "Effect of low oxygen tension on the biological characteristics of human bone marrow mesenchymal stem cells." in: Cell stress & chaperones, Vol. 21, Issue 6, pp. 1089-1099, (2016) (PubMed).

Hayashi, Hsiao, Sami, Lancero, Schlieve, Nguyen, Yano, Nagahashi, Ikeya, Matsumoto, Nishimura, Fukuda, Hisatake, Tomoda, Asaka, Toguchida, Conklin, Yamanaka: "BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, Issue 46, pp. 13057-13062, (2016) (PubMed).

Ling, Duan, Ni, Xu: "2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside Promotes Expression of the Longevity Gene Klotho." in: Oxidative medicine and cellular longevity, Vol. 2016, pp. 3128235, (2016) (PubMed).

Zhang, Gong, Wang, Chen, Lim, Dolata, Chen: "Cryptosporidium parvum infection attenuates the ex vivo propagation of murine intestinal enteroids." in: Physiological reports, Vol. 4, Issue 24, (2016) (PubMed).

Lee, Qi, Bradner, Said, Doan, Forscher, Yang, Koeffler: "Synergistic effect of JQ1 and rapamycin for treatment of human osteosarcoma." in: International journal of cancer, Vol. 136, Issue 9, pp. 2055-64, (2015) (PubMed).

Gan, Dang, Han, Ling, Duan, Liu, Xu: "ERK5/HDAC5-mediated, resveratrol-, and pterostilbene-induced expression of MnSOD in human endothelial cells." in: Molecular nutrition & food research, (2015) (PubMed).

Lee, Yi, Ahn, Lim, Hong, Cho, Lim, Song, Kwon: "Senescing human bone-marrow-derived clonal mesenchymal stem cells have altered lysophospholipid composition and functionality." in: Journal of proteome research, Vol. 13, Issue 3, pp. 1438-49, (2014) (PubMed).

Du, Klein, Hassounah, Zhang, Zhang, Wang: "Aging increases CCN1 expression leading to muscle senescence." in: American journal of physiology. Cell physiology, Vol. 306, Issue 1, pp. C28-36, (2014) (PubMed).

Tabibian, OHara, Splinter, Trussoni, LaRusso: "Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis." in: Hepatology (Baltimore, Md.), Vol. 59, Issue 6, pp. 2263-75, (2014) (PubMed).

Ryu, Park, Kim, Choi, Nam: "Protective effect of porphyra-334 on UVA-induced photoaging in human skin fibroblasts." in: International journal of molecular medicine, Vol. 34, Issue 3, pp. 796-803, (2014) (PubMed).

Tabibian, Trussoni, OHara, Splinter, Heimbach, LaRusso: "Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis." in: Laboratory investigation; a journal of technical methods and pathology, Vol. 94, Issue 10, pp. 1126-33, (2014) (PubMed).

Choo, Tai, Hymavathee, Wong, Nguyen, Huang, Cheong, Kamarul: "Oxidative stress-induced premature senescence in Wharton's jelly-derived mesenchymal stem cells." in: International journal of medical sciences, Vol. 11, Issue 11, pp. 1201-7, (2014) (PubMed).

Oishi, Imai, Go, Imamura, Hirai, Takada: "Sporadic premature aging in a Japanese monkey: a primate model for progeria." in: PLoS ONE, Vol. 9, Issue 11, pp. e111867, (2014) (PubMed).

Target Details

Background: Normal primary cells proliferate in culture for a limited number of population doublings prior to undergoing terminal growth arrest and acquiring a senescent phenotype. This finite life span correlates with the age of the organism and with the life expectancy of the species from which the cells were obtained, such that the older the age or the shorter the life span, the less the ability of the cells to undergo population doubling. Senescent cells are characterized by an irreversible G1 growth arrest involving the repression of genes that drive cell cycle progression and the upregulation of cell cycle inhibitors like p16INK4a, p53, and its transcriptional target, p21CIP1. They are resistant to mitogen- induced proliferation, and assume a characteristic enlarged, flattened morphology. Research into the pathways that positively regulate senescence and ways cells bypass senescence is therefore critical in understanding carcinogenesis. Normal cells have several mechanisms in place to protect against uncontrolled proliferation and tumorigenesis. Senescent cells show common biochemical markers such as expression of an acidic senescence- associated ß-galactosidase (SA-ß-Gal) activity. While senescence has been characterized primarily in cultured cells, there is also evidence that it occurs in vivo. Cells expressing markers of senescence such as SA-ß-Gal have been identified in normal tissues. The Cellular Senescence Assay Kit provides an easy-to-use and efficient method to determine cellular senescence. SA-β-galactosidase catalyzes the hydrolysis of X-gal, which produces a blue color. Each kit provides sufficient quantities to perform up to 50 assays in 35 mm wells.