Figure 5.

Label-free angiogenesis assays to confirm the phenotype of CRISPR-Cas9 gene-edited TERT2-HUVECs measured using the PhaseFocus Livecyte

Wild-type HUVECs, wild-type TERT2-HUVECs, and CRISPR/Cas9 gene-edited TERT2-HUVECs expressing HiBiT E-selectin (clone C8) were seeded at 20,000 cells/well onto black, flat bottomed plates pre-coated with 60 μL/well Geltrex. Cells were immediately placed within the humidified LivecyteCell Imaging chamber (Phasefocus) maintained at 37°C 5% CO₂ in air and left to equilibrate for 20 min.

(A) A single 1 mm² region per well was imaged with a 10× objective every hour for 10 h. Integrated angiogenesis network analysis was performed using PhaseFocus Cell Analysis Toolbox version 3.8.1 on each well over the total length of the assay. Images shown are for the 10 h time point.

(B) Example of the analysis with individual cells indicated by multicolored shapes and tube networks shown in yellow.

(C) The total length of the network (μm) was calculated on a per cell basis at the 10 h time point. Data are expressed as the mean ± S.E.M of 4 independent experiments (4–5 wells analyzed per cell line, per experiment).