Neutralized rat tail collagen type 1

Endothelial cell monolayer disruption (transmigration) by monocytes +/− LPS and TLR2 ligand stimulation was measured using the 9600Z Electrical Cell-Substrate Impedance Sensing (ECIS) system (Applied BioPhysics, Troy, NY, USA). A 96-well plate containing 20 gold film electrodes per well (ibidi) was coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific) for 10 min at room temperature and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific) for 45 min at 37 °C and 5% CO₂. Wells were then inoculated with LECs at a seeding density of 30,000 cells per well in 100 µL huMEC complete media at 37 °C and 5% CO₂. The run was performed under a single frequency of 4000 Hz and continuously monitored every 60 s. Impedance (Z) was determined by Ohm’s law: Z = V/I. After 20 h, when an endothelial monolayer was reached, KO and WT THP-1 cells with and without stimulants were added at a density of 100,000 cells per well in 50 µL huMEC complete media. For stimulation, 100 ng/mL LPS (Thermo Fisher Scientific) or 300 ng/mL of either TLR2-specific ligand Pam2CSK4 (Pam2) or Pam3CSK4 (Pam3) (both InvivoGen) was used. Endothelial monolayer disruption was observed for 10 h.

An EC monolayer breakdown (transmigration) assay was performed using the electrical cell-substrate impedance sensing (ECIS) model 9600Z (Applied BioPhysics, Troy, NY, USA). Opto-TLR4-LOV LECs, opto-TLR4 ΔECD2-LOV LECs and opto-TLR4-LOV HUVECs were seeded at a density of 40,000 cells/100 µL of complete medium onto a 96-well plate containing 20 gold film electrodes per well (96W20idf PET; ibidi, Gräfelfing, Germany; 72098) pre-coated with 1 mg/mL neutralized rat tail collagen type I (Thermo Fisher Scientific, Vienna, Austria; A1048301) at room temperature for 10 min, and 2 µg/mL bovine plasma fibronectin (Thermo Fisher Scientific, Vienna, Austria; 33010018) at 37 °C for 45 min. A small-amplitude AC signal (4000 Hz) (I) was imposed across the electrodes, onto which cells were attached, resulting in a potential (V) across the electrodes that was measured using the ECIS instrument [24 (link)]. The impedance (Z) was determined by Ohm’s law Z=V/I. ECs were grown to confluent monolayers for 27 h before being treated with blue light, LPS (100 ng/mL), 50,000 c/well THP-1, 50,000 c/well THP-1 M0, 50,000 c/well PBMC, combinations of blue light or LPS, and the mentioned cell types, or left untreated. EC breakdown was assessed by continuous resistance measurement for 3 h.