All HeLa cells were cultured in MEM Eagle (Sigma, US) complemented with 10% FCS (PAN Biotech, D), 1% Pen/Strep, 1% L-Glutamine, and 1% MEM NEAA (all Gibco, US). They were mycoplasma negative as tested on a trimestral basis using the MycoProbe Mycoplasma Detection Kit CUL001B. RPE-1 cells were grown in complete Dulbeccos MEM (DMEM, Sigma) at 37 °C supplemented with 10% foetal bovine serum (FBS), 2 mM L-Glutamine, penicillin and streptomycin. For transfection, cells were dissociated using trypsin and plated in tissue culture dishes (Falcon, US). After 24 h, the medium was changed and the cells were transfected using Fugene for plasmids (Promega, US) or INTERFERin (Polyplus, F) for silencing with siRNA. The cells were incubated for 24 h to 48 h (for plasmids) or 72 h (for siRNA) before performing experiments. Drug treatments were used at: nocodazole (2 h at 10 µg/mL), Taxol (4 h at 5 µg/mL) both in IM medium (described in 3H-labelling). Taxol treatments for IF were done in complete medium. ML348 and ML349 were used at 10 μM in complete medium for 4 h of pre-treatment followed by the indicated time before harvest. Tunicamycin was used at 10 μg/ml for 4 h in complete medium.
Fugene for plasmids
Manufactured by Promega
Sourced in United States
FuGENE is a transfection reagent designed to efficiently deliver plasmid DNA into a variety of cell types. It is a proprietary, multi-component lipid-based formulation that forms complexes with DNA, which are then taken up by cells.
Automatically generated - may contain errors
2 protocols using fugene for plasmids
1
HeLa and RPE-1 cell culture and transfection
2
HeLa and RPE-1 cell culture and transfection
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All HeLa cells were cultured in MEM Eagle (Sigma, US) complemented with 10% FCS (PAN Biotech, D), 1% Pen/Strep, 1% L-Glutamine, and 1% MEM NEAA (all Gibco, US). They were mycoplasma negative as tested on a trimestral basis using the MycoProbe Mycoplasma Detection Kit CUL001B. RPE-1cells were grown in complete Dulbeccos MEM (DMEM, Sigma) at 37°C supplemented with 10% foetal bovine serum (FBS), 2 mM L-Glutamine, penicillin and streptomycin. For transfection, cells were dissociated using trypsin and plated in tissue culture dishes (Falcon, US). After 24 h, the medium was changed and the cells were transfected using Fugene for plasmids (Promega, US) or INTERFERin (Polyplus, F) for silencing with siRNA. The cells were incubated for 24 h to 48 h (for plasmids) or 72 h (for siRNA) before performing experiments. Drug treatments were used at: nocodazole (2 h at 10 µg/mL), or Taxol (4 h at 5 µg/mL) both in IM medium (described in 3 H-labelling). Taxol treatments for IF were done in complete medium. ML348 and ML349 were used at 10 µM in complete medium for 4 h of pre-treatment followed by the indicated time before harvest.
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