M 352

Clonogenic assays were performed in 6-well plates that were pre-coated with 500 μl of 12 mg/ml poly (2-hydroxyethyl methacrylate) (Sigma, St. Louis, MO) in 95% ethanol. The plates were allowed to dry at 37°C. Wells were first overlaid with 2 ml of 1.5% methyl cellulose (Fisher, M-352, Pittsburgh, PA) in NSA (MCNSA), and then incubated for 1 h at 37°C before plating. For each well, cells were resuspended with 1.5% MCNSA at a 1:3 ratio in a total volume of 2 ml. Cells were fed 0.5 ml of 1.5% MCNSA every 2 to 3 days. Subsequent sphere formation was monitored and scored by light microscopy after 10 to 13 days. Five random fields at 4x magnification were photographed in triplicate for each condition and all measurements were performed using a Motic AE31 light microscope equipped with a Moticam 2300 camera and analyzed using the Motic Images Plus 2.0 ML software (Motic Instruments, Richmond BC, Canada) as previously described [43 (link)].

Sphere-formation assays were carried out in DMEM/F12 medium (11330-032, Fisher Scientific) supplemented with 20 ng/mL FGF-Basic (PHG0026, Fisher Scientific), 1× B27 supplement (12587010, Fisher Scientific), and 0.75% methylcellulose (M-352, Fisher Scientific). Cells were seeded into 96-well ultra-low attachment plates (3474, Corning) at a density of 100 cells/well. LIF was added at 250 ng/mL (12 nM). eLIFR-Fc or hLIFR-Fc was added at 25, 150, or 500 nM. Cells were allowed to grow as spheres for 1–2 weeks before each whole well (4× zoom) was imaged on an Incucyte (Satorius). In ImageJ (1.49 v), spheres were identified by shape and size, circled, and the total area of spheroids in each well was calculated. Experiments were repeated over six wells in each condition.