Fluoroblok

IL-33-induced motility of HUVECs was assessed using FluoroBlok (BD Biosciences) (8-μm pore size). Briefly, the lower surface of the filter was coated with 10 μg of gelatin. M199 medium containing 1% BSA and rIL-33 was placed in the lower wells. HUVECs were suspended at a final concentration of 10⁶ cells per ml in M199 medium containing 1% FBS. One hundred microlitres of the cell suspension was loaded into each of the upper wells, and the chamber was incubated at 37 °C for 4 h. Cell migration was evaluated by counting cells that migrated to the lower side of the filter under a confocal microscope (× 200) after staining with 1 μM Calcein AM.

Monocytes were treated with PARPi, labeled as described above, and washed before addition to BMVEC. FluoroBlok (BD Bioscience, Bedford, MA) cell culture inserts, intended to block the transmission of fluorescent light between 490 and 700 nm, were used to permit continuous detection of fluorescently labeled monocytes migrating across endothelial monolayers. BMVEC were seeded on collagen type I coated 3-μm pore 24-well tissue culture FluoroBlok inserts at a density of 2.5 × 10⁴ cells per insert. Confluent monolayers were then exposed to TNFα (20 ng/ml) for 24 h to activate the BMVEC. After activation, BMVEC were rinsed with fresh medium and the medium was replaced. For migration assays, calcein-AM-labeled monocytes were added to the upper chamber of the tissue culture insert system, while the chemoattractant, CCL2/MCP-1 (30 ng/ml), was added to the lower chamber to stimulate migration. Chemotaxis towards MCP-1 was allowed for 2 h as described [8 (link)]. The number of migrated monocytes was determined using ImageJ software (NIH) and presented as fold difference in migration from triplicate determinations, calculated from the number of migrated monocytes for each experimental condition divided by the number of migrated monocytes in the untreated, no chemoattractant control (assigned a value of 1, equivalent to 37 migrated cells).

Endothelial cells (50,000 HUVEC or 35,000 BOEC) were seeded in FN‐coated 24‐well cell culture inserts (Corning FluoroBlok, Falcon, 3.0‐μm pore size 351151) in a 24‐well plate (Corning Companion Plate, Falcon, 353504) and cultured for 48 h. Endothelial cells were treated with 10 ng/ml TNFα 20 h before the experiment. 100,000 DiO labeled neutrophils (1:6,000) and 100 μg Texas‐Red‐Dextran (70 kDa; Sigma) in HEPES medium (20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM CaCl₂, 1 mM MgSO₄, 1.2 mM K₂HPO₄, 5 mM glucose (All Sigma‐Aldrich), and 0.4% (w/v) human serum albumin (Sanquin Reagents, Amsterdam, The Netherlands), pH7.4) were added to the upper compartment of the culture insert in a total volume of 120 μl. 0.1 nM C5a (Sigma C‐5788) in HEPES medium was added to the bottom compartment in a total volume of 600 μl. Leakage and neutrophil TEM were measured simultaneously for 20 min with an interval of 1 min using an Infinite F200 pro‐plate reader (TECAN) at 37°C. DiO‐labeled neutrophil TEM dynamics were measured using EX BP 490/9 and EM BP 535/20. Leakage dynamics of Texas‐Red‐Dextran were measured with EX BP 595/9 and EM BP 630/20. To measure basal leakage, just Texas‐Red‐Dextran was added to the upper compartment.

Cell invasion assay was carried out using the BD Falcon^™ BioCoat tumour invasion systems (Corning, Arizona, USA) with fluoroBlock^™ 96 well insert plate according to manufacturer’s guidelines. This was also compared with migration of cells through uncoated BD Falcon™ FluoroBlok^™ 96 well insert plates (see Balahmar et al. 2018 (link)) for details). In this study, spheroid cells were used that have a total diameter above 8.0 μm. Therefore, the spheroids were disintegrated by using trypsin into single cells before performing the invasion assay. The number of cells invaded from the tumour invasion plate together with percentage invasion (see below for equation) was analysed using Image J software. $$\text{Invasion}\%=\frac{\text{Number of cells invaded}}{\text{Number of cells migrated}}\times 100.$$

ECs (n=200,000) were cultured in FN-treated 24-well cell culture inserts (Corning FluoroBlok, Falcon, 3.0-μm pore size # 351151) and treated with TNF-α overnight. 30 μg FITC–dextran (70 kDa; Sigma) in HEPES medium (20 mM HEPES, 132 mM NaCl, 6 mM KCL, 1 mM CaCL₂, 1 mM MgSO₄, 1.2 mM K₂HPO₄, 5 mM glucose (all from Sigma-Aldrich), and 0.4 % (w/v) human serum albumin (Sanquin Reagents), pH7.4) was added to the upper and 0.1 nM C5a (Sigma C-5788) in HEPES medium was added to the lower compartment. FITC–dextran and calcein red–orange (Molecular probes C34851) labelled neutrophil (200,000 cells) extravasation was monitored simultaneously for a period of 60 min with an interval of 1 min by an Infinite F200 pro plate reader (TECAN) at 37 °C. EX BP 485/9 and EM BP 535/20 was used to measure FITC–dextran kinetics. EX BP 535/9 and EM BP 595/20 was used to measure neutrophil (calcein red–orange) transmigration kinetics.