Culture of Cos7 cells was performed as previously described (6 (link),8 (link)). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 8% heat inactivated FBS and kept in a 37°C humidified 5% CO2 incubator. Cells were seeded into 96-well plates at a density of 15,000–20,000 cells per well (determined using a Countess Counter™, Invitrogen) one day prior to transfection. Cells were transiently transfected using polyethylenimine as described previously (6 (link),8 (link)). Hemagglutinin-tagged human CLR, hemagglutinin-tagged human CTR (CT(a) subtype) and myc-tagged human RAMP1 in pcDNA3 or 3.1 plasmid vectors were under the control of the human cytomegalovirus promotor and used as previously described (6 (link),7 (link)).
Protocol detail
Transient Transfection of COS7 Cells
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Cells
Cells culture
Cultured medium
Eagle
Hemagglutinin
Human
Human cytomegalovirus
Plasmid
Polyethylenimine
Transfection
Vectors
Corresponding Organization : University of Auckland
Other organizations : University of Iowa, University of Birmingham
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Variable analysis
independent variables
- Transfection of Cos7 cells with hemagglutinin-tagged human CLR, hemagglutinin-tagged human CTR (CT(a) subtype), and myc-tagged human RAMP1 in pcDNA3 or 3.1 plasmid vectors
- Not explicitly mentioned
- Culture of Cos7 cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 8% heat inactivated FBS, kept in a 37°C humidified 5% CO2 incubator
- Seeding of Cos7 cells into 96-well plates at a density of 15,000–20,000 cells per well one day prior to transfection
- Positive control: Hemagglutinin-tagged human CLR, hemagglutinin-tagged human CTR (CT(a) subtype), and myc-tagged human RAMP1 in pcDNA3 or 3.1 plasmid vectors (as described in the references 6, 7, and 8)
- Negative control: Not explicitly mentioned
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