Quantitative Cellular Senescence Assay

Produktnummer ABIN2344917

Produktdetails

Reaktivität: Säugetier

Nachweismethode: Fluorometric

Applikation: Quantification (Q)

Proben: Cell Samples

Produktmerkmale: Quantitative Cellular Senescence Assay Kit uses a fluorogenic substrate to measure SA-ß-Gal activity. This compound is a membrane permeable, non-fluorescent substrate of ß-galactosidase, which after hydrolysis of the galactosyl residues emits green fluorescence and remains confined within the cell. In comparison to the conventional cytochemical assay using X-gal, the use of a fluorogenic substrate greatly enhanced the sensitivity of the assay. Also, the non-toxic fluorogenic substrate allows you to study living cells by flow cytometry for more quantitative measurement of SA-ß-Gal activity. Each kit provides sufficient quantities to perform up to 10 assays in 35 mm wells.

Bestandteile:

1. Cell Pretreatment Solution (1000X) : One 25 μL tube
2. SA-ß-Gal Substrate (200X) : One 100 μL amber tube 2

Benötigtes Material:

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

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Uses a fluorogenic substrate to measure senescence-associated ß-galactosidase activity
• Assay performed in a standard 35 mm culture dish

Aufbereitung der Reagenzien:

• 1X Cell Pretreatment Solution: Prepare desired amount of 1X Cell Pretreatment Solution by diluting the provided 1000X stock 1:1000 in culture medium. For example, add 4 μL of 1000X Cell Pretreatment Solution to 4.0 mL of culture medium. Use the diluted cell pretreatment solution within 4 hrs.

Testdurchführung:

1. Aspirate the medium from the senescent cells expressing SA-ß-Gal and add 2 mL of 1X Cell Pretreatment Solution. Incubate at 37 °C for 2 hrs.
2. Add 10 μL of 200X SA-ß-Gal Substrate Solution directly to the cells in 1X Cell Pretreatment Solution. Gently mix and incubate at 37 °C for 4 hrs to overnight.
3. Wash the stained cells three times with 3 mL of 1X PBS.
4. Analyze the senescent cells by one of the following methods a. Flow cytometer after cells are trypsinized and washed in cold PBS containing 2 % FBS. b. Epifluorescence microscope (Excitation: 485 nm/Emission: 520 nm)

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: -20 °C

Informationen zur Lagerung: Aliquot and store all components at -20°C.

Referenzen

Hu, Sung, Jessen, Thibault, Finkelstein, Khan, Sacaan: "Mechanistic Investigation of Bone Marrow Suppression Associated with Palbociclib and its Differentiation from Cytotoxic Chemotherapies." in: Clinical cancer research : an official journal of the American Association for Cancer Research, (2016) (PubMed).

Chae, Park, Park, Lee, Park: "Gardenia jasminoides extract-capped gold nanoparticles reverse hydrogen peroxide-induced premature senescence." in: Journal of photochemistry and photobiology. B, Biology, Vol. 164, pp. 204-211, (2016) (PubMed).

Won, Lee, Choi, Ha, Kim, Kim, Kim, Yu, Seo, Kim, Cho, Kang: "Elucidation of Relevant Neuroinflammation Mechanisms Using Gene Expression Profiling in Patients with Amyotrophic Lateral Sclerosis." in: PLoS ONE, Vol. 11, Issue 11, pp. e0165290, (2016) (PubMed).

Grasso, Garcia, Hamidi, Cano, Calvo, Lomberk, Urrutia, Iovanna: "Genetic inactivation of the pancreatitis-inducible gene Nupr1 impairs PanIN formation by modulating Kras(G12D)-induced senescence." in: Cell death and differentiation, Vol. 21, Issue 10, pp. 1633-41, (2014) (PubMed).

Landowski, Gard, Pond, Pond, Nagle, Geffre, Cress: "Targeting integrin ?6 stimulates curative-type bone metastasis lesions in a xenograft model." in: Molecular cancer therapeutics, Vol. 13, Issue 6, pp. 1558-66, (2014) (PubMed).

Kim, Nakasaki, Todorova, Lake, Yuan, Jamora, Xu: "p53 Induces skin aging by depleting Blimp1+ sebaceous gland cells." in: Cell death & disease, Vol. 5, pp. e1141, (2014) (PubMed).

Antigendetails

Hintergrund: 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. Our