Dm lb2 microscope camera

Glass coverslips in 24-well cell culture plates were pre-coated with reduced growth factor Matrigel at the concentration of 18 mg/ml (354263, Corning, Bedford, US) and incubated for 30 minutes at 37°C. Melanoma cells and endothelial cells were transferred at the density of 4×10⁴ or 9×10⁴ cells per well in complete specific medium, respectively, incubated at 37°C 5% CO₂ and then were observed for their capacity to form tubule-like structures or vascular tubules.
To test the ability of scFv OC-46F2 to inhibit tubule formation by melanoma cell lines SKMEL28 transfected with empty vector pcDNA3.1 or scFv OC-46F2 or control scFv [22 (link)] were plated on Matrigel in complete specific medium and incubated at 37°C 5% CO₂ for 48 hours. Moreover, OC-46F2 or control scFv and L19-IL2 were added at the concentration of 200 μg/ml after four to six hours when SKMEL28, MeTA and TIME started to form tubules [19 (link)]. All experiments were performed in triplicate. After fixation in 2% paraformaldehyde in PBS, the tubules formed were counted on ten different high magnification microscopic fields per coverslip under light microscopy (Leica Microsystems, Wetzlar, Germany) at 100X or 50X magnification for melanoma and endothelial cells, respectively. Images were captured using a DM LB2 microscope camera (Leica).

This procedure was previously described in [25 (link)]. Human ovarian carcinoma cell SKOV3 and human adherent ovarian carcinoma cells were seeded on Matrigel at a density of 10 ⁵ cells per well in complete specific medium incubated at 37 °C with 5% CO₂, and were then observed for their capacity to form tubule-like structures. ED-B was added at a concentration of 2 g/mL after two hours when ovarian carcinoma cells started to form tubules, to test the ability of the ED-B fragment of fibronectin to induce tubule formation via SKOV3 and ovarian carcinoma cell OS13. To test the ability of OC-46F2 and anti-B-FN scFv to inhibit tubule formation via SKOV3 and OS13, antibodies were added at a concentration of 200 g/mL when ovarian carcinoma cells started to form tubules. All experiments were performed in triplicate. After fixation in 2% paraformaldehyde in PBS, the tubules formed were counted on ten different high magnification microscopic fields per coverslip under light microscopy (Leica Microsystems, Wetzlar, Germany) at 100X. Images were captured using a DM LB2 microscope camera (Leica) [53 (link)].