EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Temecula, CA, USA). ECMatrix gel solution was mixed with ECMatrix diluent buffer and placed in a μ-Slide plate at 37 °C for 1 h to allow the matrix solution to solidify. The EPCs were pre-treated as described above and then harvested with trypsin/EDTA. In total, 2 × 104 cells were placed into the wells containing the solid matrix with EGM-2 media at 37 °C for 16 h. Tube formation was observed using an inverted light microscope (×100). Three independent representative fields were assessed in each well, and the average numbers of tubes was determined.
Angiogenesis assay kit
Manufactured by Merck Group
Sourced in United States
The Angiogenesis Assay Kit is a laboratory tool used to measure and analyze the process of angiogenesis, which is the formation of new blood vessels from existing ones. It provides a quantitative assessment of this biological process.
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Lab products found in correlation
Crystal violet, by Beyotime (3 mentions)
Pth 1 34, by R&D Systems (3 mentions)
Nod cg prkdcscid il2rgtm1wjl szj nsg mice, by Jackson ImmunoResearch (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Osmi 1, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
Costar transwell, by Corning (1 mentions)
4 5 dimethylthiazol 2 yl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Annexin 5 dead cell kit, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Welgene (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Ags human gastric adenocarcinoma cells, by American Type Culture Collection (1 mentions)
15 protocols using angiogenesis assay kit
1
In Vitro Angiogenesis Assay for EPCs
2
LIGHT Enhances EPC Tube Formation
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Late EPC tube formation is important for angiogenesis. An Angiogenesis Assay Kit (Chemicon, Temecula, CA, USA) was used to evaluate the effects of LIGHT on EPC tube formation. Thawed ECMatrix gel solution was mixed with diluent and added to 96-well plates. The plates were incubated at 37 °C for 1 h for solidification. EPCs treated with LIGHT for 24 h were collected, and 103 cells were added to the solidified matrix. The cells were cultured at 37 °C for 12 h. An inverted optical microscope was used for observation. Four fields were randomly selected for photography in each well. The lumen-like structures with intact peripheries were calculated and compared the mean values of four field in each hole in EPCs treated with different concentrations of LIGHT.
3
In vitro Angiogenesis Assay for EPCs
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EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25 ]. EPCs (1 × 104) were grown in EC matrix solution with EGM-2 MV medium, after which the number of tube formation in the matrix gel was examined under a microscope after 16 h of incubation. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quantified in three random fields; the total area of tube formation represented the degree of angiogenesis.
4
Assessing EPC Neovasculogenic Capacity
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In vitro tube formation assays were performed on EPCs to assess the neovasculogenic capacity, which is believed to be important for endothelial function. Human EPCs were treated with recombinant human tumor necrosis factor alpha (TNF-α for 24 h, and FIR therapy was performed simultaneously every 8 h (3 times in 24 h). After the treatment of EPCs was completed, an angiogenesis assay kit (Chemicon, Billerica, MA, USA) was used to investigate the capability of tube formation [18 (link)].
5
Angiogenic Potential of EPCs via Tube Formation
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The tube formation assay was performed on EPCs to assess angiogenic capacity, which is believed to be important for new vessel formation. The in vitro tube formation assay was performed using the Angiogenesis Assay Kit (Chemicon, CA, USA)[38 (link)] according to the manufacturer’s protocol. Briefly, ECMatrix gel solution was thawed at 4°C overnight, mixed with ECMatrix diluent buffer, and placed in a 96-well plate at 37°C for 1 hour to allow the matrix solution to solidify. EPCs were treated with oxLDL for 24 hours and then harvested. A total of 104 cells were placed on the matrix solution, and the samples were incubated at 37°C for 12 hours. Tubule formation was inspected under an inverted light microscope, four representative fields were taken. The average of the total area of complete tubes formed by cells was compared using the Image-Pro Plus computer software.
6
Evaluating Endothelial Progenitor Cell Angiogenic Potential
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EPCs (1×10 5 ) were plated in the upper portion of Transwell chambers (8 μm, 24-well plates) that had been coated with Matrigel [24] . The insert membranes were cut out and stained with crystal violet (Beyotime Technology, China), and invading cells were photographed and counted using an inverted phase-contrast microscope.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, at 37 °C for 16 h. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, at 37 °C for 16 h. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
7
Evaluating EPC Invasion and Tube Formation
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EPCs (1×10 5 ) were plated in the upper portion of Transwell chambers (8 μm, 24-well plates) that had been coated with Matrigel [27] . The insert membranes were cut out and stained with crystal violet (Beyotime Technology, China), and invading cells were photographed and counted using an inverted phase-contrast microscope.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, after which the number of tube-formation in the matrix gel were examined under a microscope after 16 hours of incubation. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, after which the number of tube-formation in the matrix gel were examined under a microscope after 16 hours of incubation. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
8
Evaluating Endothelial Cell Angiogenesis
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EPCs (1×10 5 ) were plated in the upper portion of Transwell chambers (8 μm, 24-well plates) that had been coated with Matrigel [27] . The insert membranes were cut out and stained with crystal violet (Beyotime Technology, China), and invading cells were photographed and counted using an inverted phase-contrast microscope.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, at 37 °C for 16 h. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
Tube formation assay EPC tube formation was assessed using an in vitro Angiogenesis Assay Kit (Chemicon, Billerica, MA, USA) as described previously [25] . EPCs (1×10 4 ) were grown in EC matrix solution with EGM-2 MV medium, at 37 °C for 16 h. Tube formation was evaluated under an inverted light microscope. The area of tube formation was quanti ed in three random elds; the total area of tube formation represented the degree of angiogenesis.
9
PAEC Tube Formation Assay
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To assess PAEC tube formation in control and IPAH cultures, cells were grown using an Angiogenesis assay kit (Millipore, Boston, MA, USA) according to the manufacturer’s protocol. Briefly, uncoated 24-well (MatTek Corp., Ashland, MA, USA) were coated with 150 μL of an ECMatrix solution supplied in the kit and allowed to solidify for 1 h. PAECs were cultured in 10-cm plates, trypsinized, resuspended in medium 199, and seeded at a density of 1.8 × 105 cells/well with either VEGF-A (50 ng/mL), OGT inhibitor (OSMI-1, 25 μM, Sigma-Aldrich, St. Louis, MO, USA), or no treatment and cultured for 6 h. Tube formation was examined using an inverted phase-contrast microscope (Olympus IX71) to assess tube formation at 6 h. Five high-power fields per condition from triplicate experiments were analyzed using Image-Pro® Plus 7.0 (Media Cybernectics, Rockville, MD, USA).
10
Humanized Bone Marrow Ossicle Formation
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