We isolated ALDHhigh and CD133-positive TC71 cells by FACS (Figure 5E) and cultured 1000 cells/well of each population on ultra low attachment plates supplemented with Mesencult to compare their sphere forming ability.
Furthermore, to evaluate the cell viability of these two populations under floating culture conditions, the PANC-1 cells of these two populations cultured under 6-day floating conditions were reseeded at a density of 1000 cells/well onto six-well attachment plates supplemented with DMEM containing 10% FBS.
After seeding, the chamber slides and the microfluidic chambers were fed for the next 2 days with Neurobasal media supplemented with 10 ng/ml FGF until neuronal attachment.
Cells were transferred to culture dishes containing RPMI media supplemented with 20% FBS to facilitate cell attachment.
For the first 24 h, the cells have been maintained in GCM supplemented with 40% FBS to allow their attachment and then, this medium was changed to GCM containing 10% FBS.
All ECs were maintained in endothelial growth media (EGM-2) (Lonza) supplemented with 10% fetal calf serum (FCS) in flasks coated with attachment factor (Gibco, Paisley, UK).
EB were formed by growing hESC in suspension in a low attachment culture plate with media supplemented with 20% knockout serum replacement.
Asynchronous cells (8000) were seeded into 96-well optical plates, and following attachment, the growth medium was supplemented with DMSO (vehicle control), ATTM, or 2ME2.
UCI101 ovarian, were cultured in 6 well low attachment plates in DMEM/F12 medium supplemented with FGF 50 ug/mL; EGF 0.2 mg/mL; insulin 5g/mL.
The supernatant was aspirated, and the cell pellets were resuspended in standard CS-C medium (Sigma, St . Louis MO, USA) supplemented with endothelial cell growth factor and endothelial cell attachment factor.
200,000 ovarian cancer cells (UCI101), were cultured in 6 well low attachment plates in DMEM/F12 medium supplemented with FGF 50 μg/mL; EGF 0.2 mg/mL; insulin 5 g/mL.
