Cytoselect 96 well cell transformation assay

Soft agar colony formation assay was performed using CytoSelect 96-well Cell Transformation Assay (Cell Biolabs, Inc.), according to the manufacturer’s guidelines. Briefly, cells were seeded in 1.2% agar in 96-well plates (1000 cells/well); n = 4 for immortalized Schwann cells and n = 6 for HEK293 cells). After 14 days, phase-contrast images were obtained using the EVOS XL Core Microscope (ThermoFisher Scientific). Quantification of the number of colonies per well and colony size was performed using ImageJ as previously described^{23 (link)}.

To assess the effect of XMD8-92 on mesothelioma cell colony formation capability, H2373 mesothelioma cells were grown as monolayer culture and exposed to XMD8-92 or vehicle for 24 h. Cells were later trypsinized and seeded onto CytoSelect 96-well cell transformation assay (Cell Biolabs Inc., San Diego, CA) following the manufacturer’s protocol. Wells were monitored for colony formation after 1 week in culture and imaged by phase contrast using an Olympus IX70 microscope (Olympus, Waltham, MA) [4 (link)].

Cell viability was evaluated using the trypan blue exclusion method after treatment with Nexrutine^R (24 and 48 h). Colony formation was carried out using the CytoSelect 96-well Cell Transformation Assay according to vendor directions (Cell Biolabs, San Diego, CA). Cells were incubated at 37°C at 5% CO₂ for 7 days after Nexrutine^R treatment. Plate was read on SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA) using 485/520 nm filters.

Anchorage-independent growth of cell lines was examined using the CytoSelect 96-well Cell Transformation Assay (CBA-130, Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer’s provided data sheet. A mixture of 1.2% agar solution, 2× Dulbecco's modified Eagle medium (DMEM), and 20% FBS was placed into each well of a 96-well plate and the plate was incubated at 4°C for 30 minutes. Then, 75 μl of a cell suspension containing 5 × 10³ cells, 1.2% agar solution, 2× DMEM, and 20% FBS was placed on the semisolid soft agar layer of each well. The plate was incubated at 37°C under 5% CO₂ in a humidified incubator for 15 minutes. The culture medium for each cell line was placed in each well and the cultures were incubated at 37°C, under 5% CO₂ in a humidified incubator for 7 days. After incubation, the soft agar and cell cultures were dissolved using agar solubilization solution and lysis buffer, respectively. The lysates were stained with Cyquant solution and read using a fluorescence plate reader (infinite F200, Tecan Group Ltd. Zürich, Switzerland).