Twelve hours after transfection, the RPE cells were lethally irradiated (9,000 rads), washed with FBS-supplemented RPMI 1640 medium and plated in 96-well flat-bottomed culture plates, using enough RPE cells to obtain complete confluence.
The multimodal measurements are here illustrated by measuring live mouse embryonic fibroblasts cells (MEF) [18], cultured in Dulbecco's modified Eagle medium, and plated on quartz-bottom dishes one day prior experiment.
After electroporation, the cells were resuspended in neuronal culture medium and plated into microfluidic chambers attached to coverslips or glass bottom culture dishes that were coated with 200 μg/mL poly-D-lysine.
Finally, cells were resuspended in the desired medium and plated.
Cells were resuspended into fresh medium and plated in 96 well plates at 5000 cells/well.
Dissociated cells were suspended in growth medium and plated onto the patterned substrates at a density of 700 cells/mm2.
The mixture was gradually diluted with serum containing medium and plated on drug-resistant MEF feeder cells.
The mixtures were then diluted 1000-fold in GTB mixed with overlay medium and plated in duplicate on agar enriched with trypticase soy broth (TSB).
After electroporation, cells were washed once with pre-warmed medium and plated on 10cm dishes containing neomycin-resistant MEFs in the presence of Y-27632 (10µM).
Neurons were pelleted at 1000×g, washed in frog neuron medium, and plated onto poly-D-lysine-coated coverslips (BD Biosciences).
Sorted cells (ΔWEHI) were washed in fresh growth medium and plated for 12 hours before harvesting for chemotaxis assays and Western analyses.
