Type 1 solution from rat tail

KATO III invasiveness was investigated using the Transwell cell culture (12 mm diameter and 8.0 μm pore size; Corning Incorporated, United States). The upper chamber membranes were covered with Collagen, Type I solution from Rat Tail (Sigma-Aldrich, Darmstadt, Germany) and located in wells comprising the 10% FBS-supplemented medium. Cells were seeded at 1 × 10⁵/insert into the upper chambers in the serum-free medium. Treatment with 100 µM TatA was finalized in the upper chamber. After 24 h of incubation at 37°C in a 5% CO₂–95% air-humidified atmosphere, filters were fixed with 4% p-formaldehyde for 10 min and then with 100% methanol for 20 min. Cells on the lower surface of the filter were stained with 0.5% crystal violet solution. The cells that migrated to the lower surface were counted in 12 random fields using the EVOS light microscope (10×) (Life Technologies Corporation).

Adhesion assays were performed either in plastic 96-well plates (flat bottom) or on glass coverslips. The coating of the substrate was done with 150 μg/mL of collagen, type I solution from rat tail (Sigma), 12.5 μg/mL of recombinant Human ICAM-1/CD54 (R&D Systems), or 5 µg/cm² of human fibronectin from R&D Systems. Primary monocytes were added to the coated substrate and incubated for 2 h at 37 °C. Afterward, the cells were processed for immunofluorescence staining with Phalloidin A568 (Invitrogen), or analyzed for cell viability with the CellTiter-Glo Luminescent Cell Viability Assay (Promega).