Matrigel membrane matrix

Frozen vials of Matrigel Membrane Matrix (Fisher Scientific, USA) were thawed on ice at +4 °C overnight. Matrigel was added (50 µL/well) into 96-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and allowed to polymerize at 37 °C for 30 min. Meanwhile, HUVECs were detached from wells/flasks using trypsin-EDTA (Gibco, Life Technologies, Carlsbad, USA). Using basal Vasculife medium (2% Fetal Bovine Serum/FBS) with or without IL-6/sIL-6R (100 ng/mL each), suspension of HUVECs was prepared, and 10⁴ cells per well were added to the polymerized Matrigel in duplicate. The plates were placed in IncuCyte S3 Live Cell Analyses System (Sartorius AG, Göttingen, Germany) and the tube formation progress was monitored over a period of 24 h by taking pictures every hour (3 images per well). The images (10× magnification) were subsequently analyzed using Angiogenesis-Analyzer macro written for ImageJ1. The macro is available online at http://imagej.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt.

Jurkat, U937, Ramos, HEL, Reh, THP-1 and NCI-H460 cells were cultured in RPMI-1640 supplemented with 10% FBS, 1x L-glutamine and 1x penicillin/streptomycin (Thermo Fisher). HeLa and 293 T cells were cultured in DMEM (Corning) supplemented with 10% FBS and 1x penicillin/streptomycin. NTERA cells were cultured in DMEM supplemented with 20% FBS and 1x penicillin/streptomycin. A549 were cultured in F-12K Medium (Thermo Fisher) and HCT 116 cells in McCoy’s 5a Medium (Thermo Fisher), both supplemented with 10% FBS, 1x L-glutamine and 1x penicillin/streptomycin. H9 hESCs were cultured feeder-free in StemMACS iPS Brew (Miltenyi Biotec) on a Matrigel Membrane Matrix (Thermo Fisher). All cell lines were cultured at 37 °C, 5% CO₂. For sample collection, adherent cell lines were washed with PBS, dissociated by adding Accumax (Thermo Fisher) for 5 min at 37 °C and subsequently washed in their respective medium. Cells from cancer cell lines were fixed with 1.6% PFA in PBS (Electron Microscopy Sciences) for 10 min at RT and stored at −80 °C.