Evos cell imaging systems microscope

Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4 °C, and 150 μl was added to each well of a 48-well microtiter plate and allowed to solidify for 1 h at 37 °C. The wells were incubated with 3.25 × 10⁴ TIME cells plus 500 ng/ml rhAng1 either alone or with 1 μM of Ang2-BD_WT, Ang2-BD_BC5, Ang2-BD_BC6, Ang2-BD_BC10, or cRGD peptide (Merck Millipore). The cells were incubated for 16–18 h at 37 °C/5% CO₂ and then washed twice in HBSS (Hanks’ balanced salt solution; Sigma). Capillary tube formation was observed using EVOS Cell Imaging Systems microscope (ThermoFisher Scientific). Images were collected with an EVOS × 2 Objective. The total number of meshes and the number of junctions of the tubes were quantified by the analysis of digitized images of the capillary-like structures using ImageJ software and the Angiogenesis Analyzer plugin.

Serum-reduced Matrigel (10 mg/ml; BD Biosciences) was thawed overnight at 4°C, and 150 μl were added to each well of a 48-well microtiter plate and allowed to solidify for 1 h at 37°C. Wells were incubated with 3.25 × 10⁴ TIME cells with 2 μM, 4 μM and 8 μM of Ang2-BD_WT and Ang2-BD_C2.36; rhAng1, 200 ng/ml, was added as the positive control. Cells were incubated for 16–18 h at 37°C/5% CO₂. Cells were then washed twice in HBSS (Hanks’ balanced salt solution, Sigma), and capillary tube formation was observed using EVOS Cell Imaging Systems microscope (Thermo Fisher Scientific). Images were taken with EVOS 2× Objective, phase-contrast. Total length and number of junctions of the tubes were quantified by analysis of digitized images using ImageJ software and the Angiogenesis Analyzer plugin of the capillary-like structures. Tube formation assay data was analyzed with GraphPad Prism version 5.00 for Windows (La Jolla, CA, USA). Data shown is the average of triplicate experiments, and error bars represent standard error of the mean. Statistical significance was determined by column statistics and t test analysis. P value < 0.05 was considered statistically significant.