We cultured HEK293 cells (ATCC) in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO2. Cells were plated onto glass coverslips coated with poly L-lysine for 24 hr before transfection. Split cells were co-transfected with plasmids encoding pcDNA3-TRPγ or empty pcDNA3.1 (mock) with GFP (1 μg and 0.5 μg, respectively) for 6–24 hr using either Polyfect or Lipofectamine LTX reagents (Qiagen, Valencia, CA or Life Technologies, Grand Island, NY).
Lipofectamine ltx
Manufactured by Qiagen
Lipofectamine LTX is a lipid-based transfection reagent used for the efficient delivery of DNA, RNA, and other nucleic acids into a variety of cell types. It is designed to facilitate the uptake and expression of these molecules in mammalian cell culture.
Automatically generated - may contain errors
2 protocols using lipofectamine ltx
1
Transfection of HEK293 cells
2
THP-1 Macrophage Differentiation and S. aureus Infection
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Human monocytic THP-1 cell line (ATCC® TIB-202™) was maintained in RPMI 1640 Glutabio medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES buffer, 1 mM sodium pyruvate, and 50 µM β-mercaptoethanol in a humidified atmosphere at 37 °C and 5% CO2. Terminal differentiation of THP-1 to macrophages was obtained by rinsing cells twice with PBS prior to incubation with 50 nM PMA in β-mercaptoethanol-free complete RPMI 1640 medium for 48 h.
S. aureus was added to the cell culture at a multiplicity of infection (MOI) of 10. For experiments requiring an incubation time longer than 3 h, 10 µg/mL gentamicin was added to the cell culture medium to inhibit growth of extracellular bacteria.
In order to knockdown Nrf2, THP-1-derived macrophages were transfected with a pre-designed siRNA targeting Nrf2 (s9492; ThermoFisher Scientific) or a universal negative control siRNA (1027310; Qiagen) using Lipofectamine LTX following the manufacturer’s recommended protocol.
S. aureus was added to the cell culture at a multiplicity of infection (MOI) of 10. For experiments requiring an incubation time longer than 3 h, 10 µg/mL gentamicin was added to the cell culture medium to inhibit growth of extracellular bacteria.
In order to knockdown Nrf2, THP-1-derived macrophages were transfected with a pre-designed siRNA targeting Nrf2 (s9492; ThermoFisher Scientific) or a universal negative control siRNA (1027310; Qiagen) using Lipofectamine LTX following the manufacturer’s recommended protocol.
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