Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 10% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml penicillin, and 50 μg/ml streptomycin, and were maintained at 37 °C in a humidified incubator with 95% air and 5% CO2. Transient transfection was performed by using the Lipofectamine 2000 (LF2000) reagent (Invitrogen). Briefly, cells were plated onto 6- or 12-well plates (for biochemical experiments) or poly-D-lysine-coated coverslips in 24-well plates (for electrophysiological recordings) 24 hrs before transfection. The amount of CLC-1 cDNA used in each well was about 300 (for biotinylation) to 700 (for shRNA knock-down) ng/mL, and the molar ratio for co-transfection (relative to CLC-1 cDNA) ranged from 1 to 3. Various expression constructs were incubated with LF2000 reagent for 20 min at room temperature, and DNA-lipofectamine diluted in Opti-MEM (Invitrogen) was added to culture wells. After 6-hr incubation at 37 °C, the medium was changed and the culture cells were maintained in the 37 °C incubator for 24–48 hrs before being used for biochemical or electrophysiological experiments. Where indicated, drugs [cycloheximide (Sigma) or 17-AAG (Sigma)] were applied to the culture medium.
Lipofectamine 2000 lf2000 reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 2000 (LF2000) reagent is a cationic lipid-based transfection reagent designed for efficient delivery of DNA, RNA, and other macromolecules into a variety of mammalian cell lines. It facilitates the uptake of nucleic acids by forming complexes with the target molecules, which can then be internalized by the cells.
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4 protocols using lipofectamine 2000 lf2000 reagent
1
Transient Transfection of HEK293T Cells
2
CPP-Mediated miRNA Delivery in Immune Cells
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Peptides were incubated with miRNA mimic at a 17:1 CPP:miRNA molar ratio (MR) in MQwater in 1/10 of final treatment volume at room temperature (RT) for 1 h to form CPP:miRNA complexes; mixed with media and added to the cells after removing the old growth media as previously described 20 (link) . Transfections were performed at 30 nM or 100 nM (miRIDIAN microRNA Mimic Negative Control #1 and miRIDIAN microRNA hsa-miR-146a mimic; all from Thermo Fisher Scientific, USA) concentration of miRNA mimics in KCs and DCs, respectively, for 48 h or 24 h. Transfection using commercial Lipofectamine2000 (LF2000) reagent (Invitrogen, USA) was conducted as a positive control following manufacturer's instructions. When indicated, KCs were stimulated with interferon-γ (IFN-γ) using 20 ng per mL concentration and DCs with lipopolysaccharide (LPS) using 1 μg per mL concentration. Cells were harvested with Qiazol (Qiagen, Germany) and kept at -80ºC until RNA extraction.
3
Transient Transfection of HEK293T Cells
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Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glutamine, 10% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml penicillin, and 50 μg/ml streptomycin, and were maintained at 37 °C in a humidified incubator with 95% air and 5% CO2. Transient transfection was performed by using the Lipofectamine 2000 (LF2000) reagent (Invitrogen). Briefly, cells were plated onto 6- or 12-well plates (for biochemical experiments) or poly-D-lysine-coated coverslips in 24-well plates (for electrophysiological recordings) 24 hrs before transfection. Various expression constructs were incubated with LF2000 reagent for 20 min at room temperature, and DNA-lipofectamine diluted in Opti-MEM (Invitrogen) was added to culture wells. After 6-hr incubation at 37 °C, the medium was changed and the culture cells were maintained in the 37 °C incubator for24 (link)25 (link)26 (link)27 (link)28 (link)29 (link)30 (link)31 (link)32 (link)33 (link)34 (link)35 (link)36 (link)37 (link)38 (link)39 (link)40 (link)41 (link)42 (link)43 (link)44 (link)45 (link)46 (link)47 (link)48 (link) hrs before being used for biochemical or electrophysiological experiments. Where indicated, drugs [MLN4924 (kindly provided by Dr. Kuo-How Huang, National Taiwan University, Taiwan), MG132 (Sigma), or cycloheximide (Sigma)] were applied to the culture medium.
4
Transient Transfection of HEK293T Cells
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Human embryonic kidney (HEK) 293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) [supplemented with 2 mM glutamine, 10% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml penicillin, and 50 μg/ml streptomycin] and were maintained at 37 °C in a humidified incubator with 95% air and 5% CO2. Transient transfection was performed by using the Lipofectamine 2000 (LF2000) reagent (Invitrogen). Cells were plated onto 12-well plates (for biochemical experiments) or poly-D-lysine-coated coverslips in 24-well plates (for electrophysiological recordings) 24 hrs before transfection. cDNA constructs were incubated with LF2000 reagent for 20 min at room temperature, and DNA-lipofectamine diluted in Opti-MEM (Invitrogen) was added to culture wells. After 6-hr incubation at 37 °C, the medium was changed and the culture cells were maintained in the 37 °C incubator for 24–48 hrs before being used for biochemical or electrophysiological experiments. Where indicated, cycloheximide (Sigma) was applied to the culture medium 24 hrs after transfection.
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