Cytodex 3 beads

The HUVEC fibrin gel bead assay was performed as described (Nakatsu & Hughes, 2008 ). Briefly, EGM-2 complete medium was used to culture HUVECs 1 day before coating onto Cytodex 3 beads (Cat # C3275, Amersham Pharmacia Biotech, Erie, PA). About 400 HUVECs per bead were added to the FACS tube and were inverted to mix every 20 min for 4 h, followed by incubating overnight in T25 flasks. The next day, 500 cell-coated beads per well were mixed with fibrin gel composed of 2-μg·ml⁻¹ fibrinogen (Sigma, F3879), 22-μ·ml⁻¹ aprotinin (Sigma, A4529) and 0.625-U·ml⁻¹ thrombin (Sigma, T6884). After clotting, 4000 HPFs in 1-ml EGM-2 complete medium containing the TAT-Sc or TAT-ANK were added to each fibrin gel. Medium containing SC-ANK or ANK was changed every day. The inverted microscope and ImageJ software were used to photograph and quantify sprouts. For isolating the mRNA of HUVECs in the fibrin gel, 3- and 4-mg·ml⁻¹ trypsin (Sigma, T1426) were used to remove the pulmonary fibroblasts and fibrin gel, respectively. After complete digestion, the cell pellets from HUVECs from 12-well plate were resuspended in the TRIzol for RNA extraction.

Fibrin gel bead assay for the study of angiogenic sprouting was performed as described previously.^{19 (link)} In brief, Cytodex-3 beads (Amersham, Buckinghamshire, UK) were hydrated in phosphate buffered saline (PBS) and sterilized by autoclaving. ECs were trypsinized and mixed with beads at concentration of ~400 ECs per bead in 1 mL of warm EC growth medium in a fluorescence-activated cell sorting tube. The mixture was incubated for 4 hours at 37°C, with shaking the tube every 20 minutes. After that, the coated beads were transferred to a 6-well culture plate in 2 mL of EC growth medium and leave for 2 hours. After washed with EC growth medium, the coated beads were mixed with 2.0 mg/mL fibrinogen (Sigma-Aldrich, Munich, Germany) solution containing 0.15 U/mL of aprotinin (Sigma-Aldrich) and 0.625 U/mL of thrombin (Sigma-Aldrich) and then, added to each well of a 24-well plate. The plate was left for 5 minutes in the hood, and then, placed in the 37°C incubator for 10 minutes to generate a clot. Fibroblasts were then seeded on top of fibrin gel at a concentration of 20,000 cells per well. A total of 1 mL of EC growth medium per well was finally added. The media was changed every other day.