Bz x700 bright field fluorescent microscope

One day prior to tube formation assay, reduced growth factor Matrigel (VWR, #47743-718) was thawed overnight at 4°C and contact inhibited HPMECs were serum-starved overnight in 0.5% fetal bovine serum (FBS). Undiluted Matrigel was added (65 μL) to each well of a 96 well plate and allowed to solidify at 37°C for 1 hour. HPMECs were then trypsinized, counted, and 1 x 10⁴ cells/well were added after mixing cells with 10% patient serum (triplicate). HPMECs were then incubated at 37°C for 4 hours. Tube formation was visualized using Keyence BZ-X700 bright-field/fluorescent microscope (Japan). Images were empirically obtained from the center of each well and analyzed with ImageJ using the “Angiogenesis Analyzer” function to quantify number of nodes, number of tubes, and tube length. The average of each sample (triplicate) was used for statistical analysis and is reported relative to control (HPMEC treated with complete growth medium).

HPMECs were grown to confluence in a 24-well plate and then serum starved overnight in 0.5% FBS. The confluent monolayer was scratched with a sterile P200 pipette tip to create a zone free of HPMECs. The media was then gently aspirated and replaced with 10% patient serum in triplicate. Images from each well were obtained using Keyence BZ-X700 bright-field/fluorescent microscope immediately after replacing media (time (t) = 0 hours) and after 8-hour incubation at 37°C with 10% patient serum (t=8h). Migration was quantified using ImageJ where the denuded area at t=8h was subtracted from the denuded area at t=0h to calculate percent area closed. The average of each sample (triplicate) was used for analysis.