Endothelial Tube Formation Assay (In Vitro Angiogenesis Assay)
(36 references)-
- 检测方法
- Fluorometric
- 应用范围
- Biochemical Assay (BCA)
- 样品类型
- Cell Samples
- 产品特性
- Endothelial Tube Formation Assay Kit utilizes ECM (extracellular matrix) gel prepared from Engelbreth-Holm-Swarm (EHS) tumor cells. Endothelial tube formation on ECM gel closely mimics the in vivo environment and may be used to test angiogenesis inhibitors before in vivo testing. Endothelial Tube Formation Assay Kit provides a robust system to assess angiogenesis in vitro. Tube formation can be achieved within 18 hours. Following staining the tube with the provided fluorescence dye, the extent of tube formation, such as average tube length and branch point, can be quantified through imaging software. Each kit provides sufficient quantities to perform up to 50 assays in 96-well plates.
- 组件
-
- ECM Gel Solution : Five 0.5 mL tubes, Ready-To-Use
- 10X Staining Buffer : One 1 mL tube
- Staining Dye : One 50 μL tube of 1 mM Calcein AM in DMSO
- 试剂未包括
-
- Endothelial cells such as HUVEC, HMEC
- Endothelial cell culture medium
- 1XPBS
- 37 °C Incubator, 5 % CO2 atmosphere 2
- Light microscope
- Fluorescence microscope
- (optional) Tube quantification software
-
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
-
- Assesses angiogenic tube formation in vitro
- Uses an ECM matrix gel
- Resembles in vivo basement membrane environment
- 试剂准备
-
- ECM gel: Thaw overnight on ice or in a frost-free 4 °C refrigerator, store the thawed ECM gel solution up to 10 days, do not refreeze. The ECM gel solution will gel rapidly at room temperature. The tube MUST be kept on ice all the time.
- 1X Staining Buffer: Prepare a 1X Staining Buffer by diluting the provided 10X stock 1:10 in dH2O. Store the diluted solution at room temperature.
- 1X Staining Solution: FRESHLY prepare 1X Staining Buffer by diluting the provided Staining Dye to 1 μM with 1X Staining Buffer. Store 1X Staining Solution at room temperature.
- 实验流程
-
I. Tube Formation
- Thaw ECM gel and prepare 1X Staining Buffer as described above.
- Add 50 μL of thawed ECM gel solution to each well of a pre-chilled 96-well sterile plate. Note: Undiluted ECM gel solution is very viscous and quickly gels at room temperature, so it MUST be kept on ice all the time. When pipetting, use a pre-chilled pipette tip to prevent any ECM gel formation during transfer.
- Incubate 30 minutes to 1 hr at 37 °C to allow the ECM solution to form a gel.
- Harvest endothelial cells and resuspend in desired culture medium containing 0.5-10 % serum and your desired angiogenesis mediators at 1-2 x 105 cells/mL.
- Add 150 μLof cell suspension (1.5-3 x 104 cells) per well onto the solidified ECM gel. Incubate the assay plate at 37 °C for 4 to 18 hrs.
- Examine the endothelial tubes using light microscope in high magnification field. It is possible to visually estimate the extent of tube formation by inspecting the overall tube length and branch points.
II. Fluorescence Labeling
- Carefully remove medium by gently blotting on paper towels. Be carefully not to disturb endothelial tubes.
- Gently wash with 100 μL of 1X Staining Buffer and remove the wash as described in step
- 3. Add 50 μL of 1X Staining Solution per well and incubate 30 min at 37 °C.
- Gently wash with 100 μL of 1X PBS and remove the wash as described in step
- Repeat this step twice. 3
- Examine endothelial cells and tubes using a fluorescence microscope. Acquire several images per well and process them using imaging analysis software such as NIH Image or Image Pro Plus. Note: Calcein AM-labeled tubular structure is stable for only 1-2 hrs in 1X PBS at 4 °C
- 限制
- 仅限研究用
-
- 储存条件
- -20 °C
- 储存方法
- Store all components at -20°C.
-
-
: "Periodontal tissue regeneration using the cytokine cocktail mimicking secretomes in the conditioned media from human mesenchymal stem cells." in: Biochemical and biophysical research communications, Vol. 484, Issue 1, pp. 100-106, (2017) (PubMed).
: "A defined mix of cytokines mimics conditioned medium from cultures of bone marrow-derived mesenchymal stem cells and elicits bone regeneration." in: Cell proliferation, Vol. 50, Issue 3, (2017) (PubMed).
: "Role of Protein Phosphatase 1 in Angiogenesis and Odontoblastic Differentiation of Human Dental Pulp Cells." in: Journal of endodontics, Vol. 43, Issue 3, pp. 417-424, (2017) (PubMed).
: "Forkhead box protein k1 recruits TET1 to act as a tumor suppressor and is associated with MRI detection." in: Japanese journal of clinical oncology, (2016) (PubMed).
: "Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells." in: Acta odontologica Scandinavica, Vol. 74, Issue 4, pp. 298-306, (2016) (PubMed).
: "Magnetic nanocomposite scaffolds combined with static magnetic field in the stimulation of osteoblastic differentiation and bone formation." in: Biomaterials, Vol. 85, pp. 88-98, (2016) (PubMed).
: "Ultrasound-stimulated microbubble enhancement of radiation treatments: endothelial cell function and mechanism." in: Oncoscience, Vol. 2, Issue 12, pp. 944-57, (2016) (PubMed).
: "The Bromodomain BET Inhibitor JQ1 Suppresses Tumor Angiogenesis in Models of Childhood Sarcoma." in: Molecular cancer therapeutics, Vol. 15, Issue 5, pp. 1018-28, (2016) (PubMed).
: "Influence of co-culture on osteogenesis and angiogenesis of bone marrow mesenchymal stem cells and aortic endothelial cells." in: Microvascular research, Vol. 108, pp. 1-9, (2016) (PubMed).
: "Toll-like receptor 4 promotes angiogenesis in pancreatic cancer via PI3K/AKT signaling." in: Experimental cell research, Vol. 347, Issue 2, pp. 274-82, (2016) (PubMed).
: "Magnetic nanofiber scaffold-induced stimulation of odontogenesis and pro-angiogenesis of human dental pulp cells through Wnt/MAPK/NF-κB pathways." in: Dental materials : official publication of the Academy of Dental Materials, Vol. 32, Issue 11, pp. 1301-1311, (2016) (PubMed).
: "Baicalein Promotes Angiogenesis and Odontoblastic Differentiation via the BMP and Wnt Pathways in Human Dental Pulp Cells." in: The American journal of Chinese medicine, Vol. 44, Issue 7, pp. 1457-1472, (2016) (PubMed).
: "Effects of bioactive cements incorporating zinc-bioglass nanoparticles on odontogenic and angiogenic potential of human dental pulp cells." in: Journal of biomaterials applications, Vol. 29, Issue 7, pp. 954-64, (2015) (PubMed).
: "TNF-? and LPS activate angiogenesis via VEGF and SIRT1 signalling in human dental pulp cells." in: International endodontic journal, Vol. 48, Issue 7, pp. 705-16, (2015) (PubMed).
: "Effect of a stable Angiotensin-(1-7) analogue on progenitor cell recruitment and cardiovascular function post myocardial infarction." in: Journal of the American Heart Association, Vol. 4, Issue 2, (2015) (PubMed).
: "Effects of sodium tri- and hexametaphosphate on proliferation, differentiation, and angiogenic potential of human dental pulp cells." in: Journal of endodontics, Vol. 41, Issue 6, pp. 896-902, (2015) (PubMed).
: "Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells." in: International journal of oncology, Vol. 46, Issue 5, pp. 1994-2002, (2015) (PubMed).
: "Silencing of miR-195 reduces diabetic cardiomyopathy in C57BL/6 mice." in: Diabetologia, Vol. 58, Issue 8, pp. 1949-58, (2015) (PubMed).
: "Odontoblastic Differentiation, Inflammatory Response, and Angiogenic Potential of 4 Calcium Silicate-based Cements: Micromega MTA, ProRoot MTA, RetroMTA, and Experimental Calcium Silicate Cement." in: Journal of endodontics, Vol. 41, Issue 9, pp. 1524-9, (2015) (PubMed).
: "Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, potentiate the anti-angiogenic effects of bevacizumab by suppressing angiopoietin2, BiP, and Hsp90? in human colorectal cancer." in: British journal of cancer, Vol. 111, Issue 3, pp. 497-505, (2014) (PubMed).
-
: "Periodontal tissue regeneration using the cytokine cocktail mimicking secretomes in the conditioned media from human mesenchymal stem cells." in: Biochemical and biophysical research communications, Vol. 484, Issue 1, pp. 100-106, (2017) (PubMed).
-
- 背景
- Angiogenesis, or neovascularization, is the process of generating new blood vessels derived as extensions from the existing vasculature. The principal cells involved are endothelial cells, which line all blood vessels and constitute virtually the entirety of capillaries. Angiogenesis involves multiple steps, to achieve new blood vessel formation, endothelial cells must first escape from their stable location by breaking through the basement membrane. Once this is achieved, endothelial cells migrate toward an angiogenic stimulus such as might be released from tumor cells or wound-associated macrophages. In addition, endothelial cells proliferate to provide the necessary number of cells for making a new vessel. Subsequent to this proliferation, the new outgrowth of endothelial cells needs to reorganize into a three-dimensionally tubular structure. Each of these elements, basement membrane disruption, cell migration, cell proliferation, and tube formation, can be a target for intervention, and each can be tested in vitro and in vivo. Several in vivo assay systems, including the chick chorioallantoic membrane (CAM) assay, an in vivo Matrigel plug assay, and the corneal angiogenesis assay, have been developed that permit a more realistic appraisal of the angiogenic response. However, these assays are time consuming, tedious and require technical specialties. One quick assessment of angiogenesis is the measurement of the ability of endothelial cells to form three-dimensional structures (tube formation). Endothelial cells form tubes on collagen or fibrin clot coated dishes after several days.
-
