Cells for immunolabelling studies were seeded at a concentration of 2 x 10 cells/mL into Falcon 6-well tissue culture dishes containing 18 mm glass coverslips or 75 cm tissue culture flasks containing 40 mL of RPMI 1640 supplemented with 10% FBS in the absence of antibiotics.
While the DMN in the previous study was defined by the functional connectivity that was seeded at the posterior cingulated cortex (PCC), the brain regions/nodes were automatically defined by an anatomical template in the current study.
In the matrigel study, islets were seeded at the time of implantation or 3 weeks after prevascularization of the chamber.
For the cell infiltration study, cells were seeded at at 5 6 × 10 cells/mm in a volume of fibrin that filled the entire void space of the scaffold (12 mm diameter, 10 mm height).
After each time point the cells were rinsed with PBS twice, lysed in 250 µl of lysis buffer used in the RPMA experiments, and boiled for 10 min. For the inhibitor studies, HSAECs were seeded at 50,000 cells per well in a 96-well plate.
For differentiation studies, cells were seeded at confluence on the film surface.
For viability studies, cells were seeded at 2500 cells per well in a 96-well plate.
For confocal studies, cells were seeded at a density of 1 × 105 cells/ml on coverslips in 6-well plates.
For demethylation studies, cells were seeded at a density of 1 × 106 cells per 100-mm dish, and incubated for 24 hrs in a growth media.
For the expression studies, cells were seeded at 300,000 cells per 35 mm dish in their own media, allowed to attach for 24 hours, and then grown in serum free MCF-7 media for 48 hours prior to harvest.
