Synthetic siRNAs and the siRNA expression vector pGPU6/GFP/Neo were delivered to cells (typically at 40% 60% confluence) using a Lipofectin reagent (Invitrogen) according to the manufacturer's instructions.
A scratch was made through cells at 90% confluence using a 20 ul pipette tip.
In brief, cells were grown on glass cover slips to confluence using a 24 well plate.
HEK293 cells were transiently transfected with the plasmids mentioned above at 60 70% confluence using a standard calcium phosphate protocol.
Cells were cultured in 75 ml culture flasks (Biochrom, Berlin, Germany) as monolayers and harvested at 80-90% confluence using a cell-scraper (Biochrom) for further experiments.
In this context, a linear scratch was made in a fibroblast monolayer reaching confluence using a pipette tip, and fibroblast migration into the wounded area in the presence or absence of IGF1 was monitored over 24 hours.
Media were changed on a daily basis and cells passaged every 2-3 dafterftereachingng 90% confluence using a brief 0.25% trypsin and EDTA treatment for 1-2 minutes at room temperature, followed by centrifugation for 3 minutes at 1200 rpm and replated at a 1 : 2 ratio.
The FDA Electrocardiogram Warehouse is 1 example where a government resource could be used by a confluence of groups to improve the science surrounding important components of the drug approval process such as cardiac safety evaluation.
PC12 and PC12-ND6 cells were grown to 90% confluence and counted using a haematocytometer.
Cells were sub-cultured when growth reached 80 90 % confluence using trypsin EDTA solution using a 1 3 split ratio.
