Limiting dilution analysis was carried out, following 14 days’ incubation, in a 96-well format with 24 wells of each dilution; 1, 5, 10, and 20 cells/well. Cells were sorted using the BD FACS ARIA II Flow Cytometer. Limiting dilution plots and stem cell frequencies were calculated using ELDA analysis (http://bioinf.wehi.edu.au/software/elda/index.html ) (29 (link)).
Sphere-forming assays were performed as described (7 (link)), with the following modifications: 100–200 single cells were seeded per well in 96 well plates, with 6 replicates per condition. Cells were cultured in 80µl of N2 medium supplemented with 20ng/ml of EGF and/or 30ng/ml of FGF2 as specified in the text. The number of spheres with a diameter greater than 70 µm was quantified on a GelCount analyzer (Oxford Optronix) 5 days after seeding.
Sphere-forming assays were performed as described (7 (link)), with the following modifications: 100–200 single cells were seeded per well in 96 well plates, with 6 replicates per condition. Cells were cultured in 80µl of N2 medium supplemented with 20ng/ml of EGF and/or 30ng/ml of FGF2 as specified in the text. The number of spheres with a diameter greater than 70 µm was quantified on a GelCount analyzer (Oxford Optronix) 5 days after seeding.