Infinite m1000

OVCAR 3 cells (4 × 10⁴) were seeded in triplicate in black Corning^® 96 well flat clear bottom black microplates (3603). Two days later, GFP-MSLN transfected HEK 293 T cells (3 × 10⁵) were incubated in the presence or absence of anti-MSLN/ANef nanobodies (1 µM) at 4°C, 30 min in RPMI 10% FCS then added to the OVCAR-3 monolayer for 1 hr at 37°C. GFP signals were recorded at 508 nm before and after 7 washes in PBS using a fluorescent plate reader (Tecan Infinite^® M1000 - Life Technologies). The percentage of adhesion was calculated using the formula: (FAW/FBWsample)/(FAW/FBWmedium) *100 as described by Bergan et al. (20 (link)) with FAW= fluorescence after washes and FBW=fluorescence before washes. Incubation without antibody corresponds to the reference condition (n=3).

Two-dimensional migration was investigated using 8 μm FluoroBlok™ (Corning® Fluoroblok™ Cell Culture Inserts, BD biosciences). Two days before the assay, tumor cells were stained with 10 μM carboxyfluorescein diacetate succidimyl ester (CellTrace™ CFSE Cell Proliferation kit, ThermoFisher Scientific) for 20 min at 37°C. One day before, cell cycle synchronization was performed by serum starvation. The upper chambers were seeded with CFSE-labeled tumor cells (10⁵) in RPMI medium with or without bsFab (50 nM). Chemotactic gradient was created by addition of 5% FBS medium in the lower chamber. The fluorescence in the bottom chamber measured at 6 and 24 h on a fluorescent plate reader (Tecan Infinite® M1000—Life Technologies) at 521 nm corresponds to the migrating cells.
Invasion assays were performed as above using 8 μm FluoroBlock™ inserts pre-coated with 1 mg/ml Matrigel.
For each experiment, fluorescence values for the control + 5% FBS were set as 100% migration/invasion. The ratiometric results were expressed relative to this sample.