96 well flat clear bottom black microplates

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).

Cells seeded in Corning 96-well Flat Clear Bottom Black Microplates (#3603) were incubated with increasing concentrations of ricin in complete medium for the indicated time. The medium was replaced with fresh complete medium including Alamar Blue (final concentration 1:10). Then, the fluorescence (540/590 nm) was measured 1 h later by a Cytation^TM 5 cell imaging multi-mode reader (BioTek, Winooski, VT, USA). Calcein-AM in PBS (1: 2000, 5 µM) was incubated with cells for 30 min and its intracellular fluorescence (495/520 nm) was measured by Cytation 5.

Isolated cells were expanded to Passage 1 (P1) in monolayer cultures. P1 cells were then seeded at 20,000 or 10,000 cells per well in eight-well chamber slides (Nunc Lab-Tek II Chamber Slide System) and 96- well flat clear bottom black microplates (Corning, NY) respectively. Cells were serum-starved in DMEM with ITS (1X) (Thermo Fisher, Waltham, MA) for 2 hr prior to treatment with 5 μM RG-7112 (Selleck Chemicals, TX), 100 μM o-Vanillin (Sigma-Aldrich, Oakville, ON, Canada) or vehicle (DMSO (0.01%, (Sigma-Aldrich, Oakville, ON, Canada) for 6 hr. Immunocytochemistry was performed as previously described (Cherif et al., 2019 (link)). Apoptosis was detected using a commercial kit (ab176749, Abcam, Cambridge, MA) according to the manufacturer’s instructions. Photomicrographs were acquired with a fluorescent Olympus BX51 microscope equipped with an Olympus DP71 digital camera (Olympus, Tokyo, Japan).