In vitro self-renewal capacity was tested with sphere formation assays, as described previously [19 (link)]. Limiting dilution assays were performed under SC conditions at different cell densities (ranging from 1 to 1,000). The cells were incubated in hypoxic or normoxic conditions for 7 d (sphere formation assays) or 10 d (limiting dilution assays). Afterward, both sphere numbers and sizes were measured under a microscope.
3D colony-formation assays were performed using a serum-free mix of 60% SCM medium and 40% methylcellulose medium, i.e. MethoCult® H4100 (StemCell Technologies, H4100), supplemented with bFGF (Miltenyi Biotec, 130-093-841 20 ng/mL) and EGF (Biomol, 50,349.5; 20 ng/mL). After 14 d, the resulting 3D colonies were counted under a microscope.
3D colony-formation assays were performed using a serum-free mix of 60% SCM medium and 40% methylcellulose medium, i.e. MethoCult® H4100 (StemCell Technologies, H4100), supplemented with bFGF (Miltenyi Biotec, 130-093-841 20 ng/mL) and EGF (Biomol, 50,349.5; 20 ng/mL). After 14 d, the resulting 3D colonies were counted under a microscope.
