Non-patterned grids were plasma cleaned. Cells were detached from cell culture flasks using 0.05 % trypsin-EDTA and seeded on pre-treated (either patterned or non-patterned) grids in glass bottom ibidi μ-Dish 35 mm high (ibidi, Martinsried, Germany).
Cells were seeded on fibronectin micropatterned surfaces right after being passed
through a cell 40 μm pore-size cell strainer (Corning, Amsterdam,
Netherlands) at a density of 2 × 104 cells/cm2 for
HeLa and 8 × 103 cells/cm2 for RPE1 cell lines.
After seeding, grids were incubated for 1.5-2 h for HeLa cells or 20-35 min for
RPE1 cells. Next, grids were transferred to a new cell-free dish and incubated
at 37°C with 5 % CO2 to allow cell adhesion to the grids.
Transfer to a new dish was beneficial to remove cells that were non-specifically
attached to areas outside the patterns. Cells were vitrified 4-6 h post-transfer
for RPE1 cells (to attain a higher number of grid squares with single cells) or
after overnight incubation for HeLa cells.
At least 60 grids have been seeded with either HeLa or RPE1 cells obtaining reproducible results with cells settling and adhering to the micropatterned areas.
Cells were seeded on fibronectin micropatterned surfaces right after being passed
through a cell 40 μm pore-size cell strainer (Corning, Amsterdam,
Netherlands) at a density of 2 × 104 cells/cm2 for
HeLa and 8 × 103 cells/cm2 for RPE1 cell lines.
After seeding, grids were incubated for 1.5-2 h for HeLa cells or 20-35 min for
RPE1 cells. Next, grids were transferred to a new cell-free dish and incubated
at 37°C with 5 % CO2 to allow cell adhesion to the grids.
Transfer to a new dish was beneficial to remove cells that were non-specifically
attached to areas outside the patterns. Cells were vitrified 4-6 h post-transfer
for RPE1 cells (to attain a higher number of grid squares with single cells) or
after overnight incubation for HeLa cells.
At least 60 grids have been seeded with either HeLa or RPE1 cells obtaining reproducible results with cells settling and adhering to the micropatterned areas.