Harvest
Exhausted medium was retrieved from the vessel and the microcarrier-cell
suspension was washed with 40 mL of HBSS (Gibco, UK) twice. 20 mL of
TryPLETM Select (Gibco, UK) were incubated with the
microcarrier-cell suspension for 15 minutes, at an agitation of 100 rpm
in a standard incubator. To quench the dissociation reagent, 30 mL of
complete medium were added. The suspension was passed through 40 µm cell
strainers and centrifuged for 5 minutes at 400 x g. Harvested cells were
counted using the trypan blue exclusion method and were either pelleted
for RT-qPCR or plated at 6000 cells/cm2 to perform
several assays such as immunocytochemistry and NG108-15 co-culture
assay.