HEK293 cells (from American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) that contained 10% (v/v) human serum, 2 mM L-glutamine, 2 U ml−1 penicillin and 2 mg ml−1 streptomycin at 37 °C in a humidity-controlled incubator with 10% CO2. F11 cells (from American Type Culture Collection) were grown in Ham's F12 medium (Invitrogen) that contained 10% (v/v) foetal bovine serum (Invitrogen), 1.5 mM L-hydroxyproline (Sigma) and 1% Glutamax (Invitrogen), at 37 °C in a humidity-controlled incubator with 10% CO2. HEK293 and F11 cells were transfected with cDNA (1 μg, unless mentioned otherwise) encoding the protein or proteins of interest using the Mirus 293 Transfection Reagent (Mirus Corporation, Madison, USA). For TIRF experiments, HEK293 or F11 cells were reseeded on 25 mm poly-L-lysine (PLL)-coated glass coverslips after 4 h of transfection, and kept under identical culture conditions for 12–18 h before imaging. For calcium imaging and patch-clamp experiments, cells were transfected overnight and then reseeded on PLL-coated coverslips for 2–6 h before recording.
Mirus 293 transfection reagent
Manufactured by Mirus Bio
Sourced in United States
Mirus 293 Transfection Reagent is a proprietary cationic lipid-based transfection agent designed for efficient transfection of mammalian cells, particularly the human embryonic kidney 293 cell line.
Automatically generated - may contain errors
Lab products found in correlation
Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L hydroxyproline, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
2 protocols using mirus 293 transfection reagent
1
Transfection and Culturing of HEK293 and F11 Cells
2
Transient Transfection of HEK293 Cells
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Human embryonic kidney cells (HEK293) were grown in Dulbecco's modified Eagle's medium (DMEM) that contained 10% (v/v) human serum, 2 mM L-glutamine, 2 units/ml penicillin and 2 mg/ml streptomycin (Invitrogen) at 37°C in a humidity-controlled incubator with 10% CO2. HEK293 cells were transiently transfected with cDNA encoding TRPM4-GFP (in the pCAGGSM2 vector) or TRPV2-GFP (in the pCiNeo vector) using Mirus 293 Transfection Reagent (Mirus Corporation, Madison, USA). For most experiments, cells were kept in the transfection medium for 4 h, and subsequently reseeded on poly-L-lysine-coated 25-mm glass coverslips with thickness of 0.16–0,19 mm (Gerhard Menzel GmbH, Germany). Thereafter, cells were kept under the same culture conditions for an additional 12 h, allowing optimal attachment of the cells to the glass coverslip, and resulting in a total of 16 h between transfection and TIRF measurement. For the experiments shown in Supplementary Figure S1 , incubation times were adjusted, such that measurements were made after between 4 and 72 hours of total transfection time.
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