Transfections were performed with cells at 80% 90% of confluence by using of Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
The cells (Vero and MRC-5) were first grown in monolayer using the respective media at 37° C and were detached after reaching confluence by standard trypsinization protocol and inoculated into bioreactor to start the bioreactor culture.
Immunofluorescent staining reveals the activation of focal adhesion and subsequent organization of F-actin from the attached cell surfaces, which suggests the TriG microspherical substrate is favorable to ADC adhesion and therefore capable of promoting HDF proliferation to achieve confluence by turning over three times within 10 days.
GI-1 cells obtained from Riken Bio Resource Center, Japan, were cultured in monolayers to 80% confluence by maintaining in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin solution in a 5% CO2-humidified atmosphere at 37°C.
Cells were passaged at 80 90% confluence by trypsinization.
Cells were maintained and passaged upon reaching confluence by standard cell culture techniques.
Both cell lines exhibited ∼90% confluence by the time of assay.
Cultures cells were split (1∶6) when they reached 90% confluence by use of 0.05% trypsin-0.5 mM EDTA (GIBCO-BRL).
Differentiation of myoblasts into myotubes was initiated at approximately 90% confluence by switching to differentiation medium containing 2% horse serum (D0, day 0 of differentiation).
Cells were induced to differentiate at 80 90% confluence by transferring them into DMEM plus 2% horse serum (differentiation medium, [DM]), 1% Penicillin/Streptomycin and 1% Glutamax (Invitrogen).
Cells were cultured in 12-well plates and transfected at 30 40% confluence by adding HiPerFect (Qiagen, Hilden, Germany) complexed with siRNA (final concentration: 20nM) as described previously [57].
