OxiSelect™ DNA Double Strand Break (DSB) Staining Kit
(6 references)-
- 适用
- 其他
- 检测方法
- Fluorometric
- 应用范围
- Biochemical Assay (BCA)
- 品牌
- OxiSelect™
- 样品类型
- Cell Samples
- 产品特性
- OxiSelect™ DNA DSB Staining Kit is based on the phosphorylation of the histone H2A.X at serine 139 in response to DNA damaging agents which cause double strand breaks in cells that are cultured in microtiter plates. This Trial Size kit provides sufficient reagents for up to 20 stainings in a 96-well plate.
- 组件
-
- Anti-Phospho-Histone H2A.X (Ser 139) Antibody (100X) : One tube - 20 μL.
- Secondary Antibody, FITC Conjugate (100X) : One amber tube - 40 μL.
- DNA DSB Inducer (20 mM) : One tube - 50 μL of 20 mM Etoposide in methanol. 2
- 试剂未包括
-
- Cell line of interest
- 3.7 % Formaldehyde in PBS
- 90 % Methanol
- PBS
- Blocking/Antibody Incubation Buffer (1 % BSA/PBS)
- Wash Buffer (PBS containing 0.05 % Tween-20)
- Fluorescence microscope with FITC filter
-
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
-
- 应用备注
- Optimal working dilution should be determined by the investigator.
- 说明
-
- Detects phosphorylation of histone H2A.X
- See immunofluorescence in about 3 hours
- DNA double-strand break inducer included
- 试剂准备
-
- 1X Anti-Phospho-Histone H2A.X Antibody Solution: Prepare a 1X Anti-Phospho-Histone Antibody Solution by diluting the provided 100X Anti-Phospho-Histone Antibody stock 1:100 in 1 % BSA/PBS. Use the working solution immediately.
- 1X Secondary Antibody, FITC Conjugate Solution: Prepare a 1X Secondary Antibody Solution by diluting the provided 100X stock 1:100 in 1 % BSA/PBS. Use the working solution immediately.
- DNA DSB Inducer: Dilute Etoposide a minimum of 1:200 in culture medium. Vortex to homogeneity. Use the working solution immediately.
- 90 % Methanol: Dilute 100 % Methanol to 90 % with DI H2O (9:1 ratio). Store the solution at -20 °C.
- 实验流程
-
The following assay protocol is written for a 96-well format. Refer to the table below for the appropriate dispensing volumes for other plate formats. Note: using other plate formats will decrease the number of assays possible with this kit. 96-well 48-well 24-well 12-well 3.7 % Formaldehyde/PBS 100 200 400 800 (μL/well) 90 % Methanol (μL/well) 100 200 400 800 Wash Buffer (μL/well) 200 400 800 1500 Blocking Buffer(μL/well) 200 400 800 1500 1X Anti-Phospho-Histone 100 200 400 800 Antibody Solution (μL/well) 1X Secondary Antibody, FITC Conjugate 100 200 400 800 Solution (μL/well) Table
- Dispensing Volumes of Different Plate Formats. 3
I. Cell Seeding
- Harvest and resuspend cells in culture medium at 5 x 105 cells/mL. Seed 100 μL in each well of a 96-well plate and incubate overnight at 37 °C, 5 % CO2 (cells should be > 80 % confluent).
- (Optional) Aspirate the culture medium and add 100 μL/well of diluted DNA DSB Inducer, or desired DSB agent, and incubate for 1 hour at 37 °C, 5 % CO2.
II. Immunofluorescence Staining
- Carefully remove medium from the wells by tilting the plate and aspirating from the edge. Fix the cells by gently adding 100 μL of 3.7 % Formaldehyde/PBS to each well of the 96-well plate, taking care not to dislodge the cells. Incubate 10 minutes at room temperature.
- Gently wash the fixed cells once with 200 μL of 1X PBS.
- Aspirate the wells and add 100 μL of ice-cold 90 % Methanol to each well. Incubate 10 minutes at 4 °C.
- Gently wash the fixed cells once with 200 μL of 1X PBS.
- Aspirate the wells and add 200 μL of Blocking Buffer (see Materials Not Supplied section) to each well. Incubate for 30 minutes at room temperature on an orbital shaker.
- Aspirate the wells and add 100 μL of 1X Anti-Phospho-Histone Antibody Solution (see Preparation of Reagents section) to each well. Incubate for 1 hour at room temperature on an orbital shaker.
- Gently wash the wells 5 times with 200 μL Wash Buffer (PBST).
- Aspirate the wells and add 100 μL of 1X Secondary Antibody, FITC Conjugate Solution (see Preparation of Reagents section) to each well. Incubate for 1 hour at room temperature on an orbital shaker.
- Gently wash the wells 5 times with 200 μL Wash Buffer (PBST).
- Aspirate and add 200 μL 1X PBS to each well.
- View staining with a fluorescence microscope using FITC filter. 4
- Dispensing Volumes of Different Plate Formats. 3
- 限制
- 仅限研究用
-
- 储存条件
- -20 °C
- 储存方法
- Store all kit components at -20°C.
-
-
: "Cellular effects induced by 17-β-estradiol to reduce the survival of renal cell carcinoma cells." in: Journal of biomedical science, Vol. 23, Issue 1, pp. 67, (2016) (PubMed).
: "Normal human gingival fibroblasts undergo cytostasis and apoptosis after long-term exposure to butyric acid." in: Biochemical and biophysical research communications, (2016) (PubMed).
: "High resistance to X-rays and therapeutic carbon ions in glioblastoma cells bearing dysfunctional ATM associates with intrinsic chromosomal instability." in: International journal of radiation biology, Vol. 91, Issue 2, pp. 157-65, (2015) (PubMed).
: "Fullerenol protects retinal pigment epithelial cells from oxidative stress-induced premature senescence via activating SIRT1." in: Investigative ophthalmology & visual science, Vol. 55, Issue 7, pp. 4628-38, (2014) (PubMed).
: "Preclinical validation of talaporfin sodium-mediated photodynamic therapy for esophageal squamous cell carcinoma." in: PLoS ONE, Vol. 9, Issue 8, pp. e103126, (2014) (PubMed).
: "A peptide inhibitor of synuclein-? reduces neovascularization of human endometriotic lesions." in: Molecular human reproduction, Vol. 20, Issue 10, pp. 1002-8, (2014) (PubMed).
-
: "Cellular effects induced by 17-β-estradiol to reduce the survival of renal cell carcinoma cells." in: Journal of biomedical science, Vol. 23, Issue 1, pp. 67, (2016) (PubMed).
-
- 背景
- DNA double-strand breaks (DSBs) are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination. A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage. H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells resulting in discrete γ-H2AX (phosphorylated H2AX) foci at the DNA damage sites. H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells and meiotic recombination in mice. Phosphorylation of H2A at serine 139 causes chromatin decondensation and appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage.
-
