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A wound was created by scratching cells in 1.5-cm zone with sterile 200-μl pipette tip after the old medium was washed out.
While the nanoparticle/DNA in the medium was washed away at 24 h after transfection, after another 24 h, GFP signals were not obviously enhanced (data not shown).
After 1 h incubation, the medium was washed well and cultured for a further 48 h.
36 hours post transfection, medium was washed off and cells were mixed.
Residual culture medium was washed off the cells with phosphate-buffered saline.
The medium was washed with PBS and replaced with EBM-2 medium supplemented with recombinant bFGF (10 ng/ml) (Promega Corp).
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For serum activation, cells in low-serum medium were washed twice with PBS, and DMEM medium containing 10% FBS was added to the cells and incubated for 4 hours.
For starvation experiments, logarithmically growing cells from unlabeled medium were washed twice with starvation medium and incubated for 3 hr in starvation medium.
Bacteria grown overnight in NYGB medium were washed and re-suspended in water to an OD at 600 nm of 0.001.
Cell polarity was then characterized by the cell length-to-width ratio 4 hdpPSCs after 6 and 12 h culture in nonselective differentiation medium were washed with PBS and placed under the optical microscope.
PBMC in medium were washed and resuspended in PBA (phosphate buffered saline containing 0.16% BSA and 0.1% sodium-azide) to a concentration of 2 million PBMC/ml and divided in aliquots of 200 μl.
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