96-well Cellular Senescence Assay (SA β-Gal Activity)
(13 references)-
- 适用
- 哺乳动物
- 检测方法
- Fluorometric
- 应用范围
- Cellular Assay (CA)
- 样品类型
- Cell Samples
- 组件
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- 2X Cell Lysis Buffer : One 10 mL bottle
- 2X Reaction Buffer : One 10 mL bottle
- SA-ß-Gal Substrate (20X) : One 300 μL amber tube
- Stop Solution : One 25 mL bottle 2
- 试剂未包括
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- Senescent cells or tissue samples
- 37 °C Incubator
- β-mercaptoethanol
- 96-well plate suitable for a fluorescence plate reader
- 96-well Fluorometer
- Protein Assay Reagents
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Tumor Necrosis Factor alpha (TNF alpha) ELISA Kit
TNF alpha 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Interleukin 1, beta (IL1B) ELISA KitIL1B 适用: 大鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 小鼠 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
High Mobility Group Box 1 (HMGB1) ELISA KitHMGB1 适用: 人 Colorimetric Sandwich ELISA 62.5 pg/mL - 4000 pg/mL Plasma, Serum
Lipopolysaccharides (LPS) ELISA Kit适用: Various Species Colorimetric Competition ELISA 12.35 ng/mL - 1000 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Tumor Necrosis Factor alpha (TNF alpha) ELISA KitTNF alpha 适用: 人 AA 77-233 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Plasma (EDTA), Plasma (citrate), Plasma (heparin), Serum
Interleukin 6 (IL6) ELISA KitIL6 适用: 人 Colorimetric Sandwich ELISA 7.8-500 pg/mL Cell Culture Supernatant, Plasma, Serum, Tissue Homogenate, Urine
Vascular Cell Adhesion Molecule 1 (VCAM1) ELISA KitVCAM1 适用: 人 Colorimetric Sandwich ELISA 1.563-100 ng/mL Plasma, Serum, Tissue Homogenate
Matrix Metalloproteinase 2 (MMP2) ELISA KitMMP2 适用: 人 Colorimetric Sandwich ELISA 0.78 ng/mL - 50 ng/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
Interleukin 1, beta (IL1B) ELISA KitIL1B 适用: 人 Colorimetric Sandwich ELISA 15.6 pg/mL - 1000 pg/mL Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Homogenate
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- 应用备注
- Optimal working dilution should be determined by the investigator.
- 说明
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- Measure activity of senescence-associated ß-galactosidase
- Quantitative results in a fluorescence plate reader
- 试剂准备
-
1X Cell Lysis Buffer: Prepare a 1X Cell Lysis Buffer by diluting the provided 2X stock 1:2 in ddH2O. Store the diluted solution at room temperature for up to six months. Immediately before use, add proper amount of proteinase inhibitors such as PMSF. 2X Assay Buffer: Immediately before use, add β-mercaptoethanol to 2X Reaction Buffer at a final concentration of 10 mM and dilute 20X SA-ß-Gal Substrate to 1X with 2X Reaction Buffer containing 10 mM β-mercaptoethanol
- Don't store 2X Assay Buffer. Reagent 96-well 24-well 6-well 10 cm 1X Cell Lysis Buffer 100 μL 400 μL 1000 μL 1500 μL Table 1. 1X Cell Lysis Buffer Required per Well for Various Culture Plates.
- 实验流程
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- Aspirate the medium from the senescent cells.
- Wash the cells once with 200 μL of cold 1X PBS and aspirate the wash.
- Add 100 μL of cold 1X Cell Lysis Buffer (see the table above for the required amount of 1X Cell Lysis Buffer of other plate formats). Incubate at 4 °C for 5 minutes. Transfer the whole lysate to a microcentrifuge tube and centrifuge 10 minutes at 4 °C. Collect supernatant as cell lysate.
- (optional) Determine the total protein concentration of each cell lysate sample by protein assay such as Pierce's BCA protein Assay.
- Transfer 50 μL of the cell lysate to a 96-well plate. Add 50 μL of freshly prepared 2X Assay Buffer. Incubate the wells at 37 °C protected from light for 1- 3 hr.
- Remove 50 μL of the reaction mixture to a 96-well plate suitable for fluorescence measurement. Stop the reaction by adding 200 μL of Stop solution.
- Read fluorescence with a fluorescence plate reader at 360 nm (Excitation) / 465 nm (Emission). 3
- 限制
- 仅限研究用
-
- 储存条件
- RT/-20 °C
- 储存方法
- Store SA-β-gal substrate solution protected from light at -20°C. Store all other components at room temperature.
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: "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).
: "Cell culture density affects the proliferation activity of human adipose tissue stem cells." in: Cell biochemistry and function, Vol. 34, Issue 1, pp. 16-24, (2016) (PubMed).
: "Anti-senescence and Anti-inflammatory Effects of the C-terminal Moiety of PTHrP Peptides in OA Osteoblasts." in: The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 72, Issue 5, pp. 624-631, (2016) (PubMed).
: "Cryptosporidium parvum infection attenuates the ex vivo propagation of murine intestinal enteroids." in: Physiological reports, Vol. 4, Issue 24, (2016) (PubMed).
: "Cholangiocyte senescence caused by lysophosphatidylcholine as a potential implication in carcinogenesis." in: Journal of hepato-biliary-pancreatic sciences, Vol. 22, Issue 9, pp. 675-82, (2015) (PubMed).
: "Depletion of B cell CLL/Lymphoma 11B Gene Expression Represses Glioma Cell Growth." in: Molecular neurobiology, (2015) (PubMed).
: "A Novel Interaction between FLICE-Associated Huge Protein (FLASH) and E2A Regulates Cell Proliferation and Cellular Senescence via Tumor Necrosis Factor (TNF)-Alpha-p21WAF1/CIP1 Axis." in: PLoS ONE, Vol. 10, Issue 7, pp. e0133205, (2015) (PubMed).
: "Ascorbic acid provides protection for human chondrocytes against oxidative stress." in: Molecular medicine reports, Vol. 12, Issue 5, pp. 7086-92, (2015) (PubMed).
: "SIRT1 counteracted the activation of STAT3 and NF-κB to repress the gastric cancer growth." in: International journal of clinical and experimental medicine, Vol. 7, Issue 12, pp. 5050-8, (2015) (PubMed).
: "Inhibition of APE1/Ref-1 redox activity rescues human retinal pigment epithelial cells from oxidative stress and reduces choroidal neovascularization." in: Redox biology, Vol. 2, pp. 485-94, (2014) (PubMed).
: "Mesenchymal stem cells from rats with chronic kidney disease exhibit premature senescence and loss of regenerative potential." in: PLoS ONE, Vol. 9, Issue 3, pp. e92115, (2014) (PubMed).
: "COH-203, a novel microtubule inhibitor, exhibits potent anti-tumor activity via p53-dependent senescence in hepatocellular carcinoma." in: Biochemical and biophysical research communications, Vol. 455, Issue 3-4, pp. 262-8, (2014) (PubMed).
: "Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis." in: Nucleic acids research, Vol. 38, Issue 17, pp. 5718-34, (2010) (PubMed).
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: "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).
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- 背景
- 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 INK4a CIP1 inhibitors like p16 , p53, and its transcriptional target, p21 . 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 96-well Cellular Senescence Assay Kit provides an easy-to-use and efficient method to determine the cellular senescence by measuring SA-ß-Gal activity using a fluorometric substrate. This quantitative assay uses cell lysate for both SA-β-galactosidase activity determination and normalization of samples containing different cell numbers. Each Trial Size kit provides sufficient quantities to perform up to 24 assays in a 96-well plate.
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