Fibrin beads

A fibrin beads sprouting assay was conducted, as described previously, using a fibrin beads assay kit (Amersham‐Pharmacia Biotech), according the supplier's instructions.31 Briefly, HUVECs were incubated with the Cytodex 3 microcarrier beads (400 cells per bead; Sigma‐Aldrich) at 37°C overnight. The microbeads were then embedded in the fibrinogen (Sigma‐Aldrich) containing 0.625 U/mL thrombin (Sigma‐Aldrich) at a density of 100 beads/mL in a 48‐well plate, and 0.5 mL EGM‐2 medium (Clonetics) was added with lung fibroblasts (20 000 cells per well), as described earlier. The medium was changed every other day for 2 or 4 days. Images of the beads were captured under a microscope (CKX41; Olympus) with a CCD camera (DP70; Olympus), and sprouting was quantified by measuring the number and length of sprouts.

Fibrin beads sprouting assay was conducted using a Fibrin beads assay kit (Amersham‐Pharmacia Biotech, Piscataway, NJ) as described previously (Yan et al., 2016). Briefly, HUVECs were incubated with the Cytodex 3 microcarrier beads (Sigma‐Aldrich; 400 cells per bead) at 37°C overnight. The beads were then embedded in the fibrinogen (Sigma‐Aldrich) containing 0.625 U/ml thrombin (Sigma‐Aldrich) at a density of 100 beads/ml in a 48‐well plate, and 0.5 ml of EGM‐2 medium (Clonetics, Walkersville, MD) was added with lung fibroblasts (20,000 cells/well). The medium was changed every other day for 2 or 4 days. Images were taken under a microscope (CKX41; Olympus) with a CCD camera (DP70; Olympus), and sprouting was quantified by measuring the number and length of sprouts.