Fzd1/2/4/5/7/8-knockout HEK293T cells (69 (link)) were maintained in DMEM supplemented with 10% v/v fetal bovine serum. Cells were seeded into white 96-well plates (PerkinElmer) and transfected with 80 ng SuperTopFlash plasmid (laboratory of Dr. Randall Moon; Addgene plasmid #12456), 20 ng CMV-driven LacZ plasmid, and 1 ng FLAG-tagged Fzd4 or FZD5 plasmid per well using Lipofectamine 2000 according to the manufacturer’s instructions (ThermoFisher). Purified xWnt8 or Norrin (with MBP tag removed as described above for BLI) were added 24 h post-transfection and cells were incubated for 16–18 h with ligand. Cells were lysed and luciferase and β-galactosidase signals were quantified on a BioTek Synergy 2 plate reader using the Dual-Light Reporter System (ThermoFisher) according to the manufacturer’s instructions.
Protocol detail
Wnt Signaling in HEK293T Cells
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Cells
Fetal bovine serum
Fzd4
Lacz
Ligand
Light
Lipofectamine 2000
Luciferase
Plasmid
Transfection
β galactosidase
Corresponding Organization : Stanford University
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Variable analysis
independent variables
- Transfection of Fzd4 or FZD5 plasmid
- Addition of purified xWnt8 or Norrin ligand
- Luciferase signal
- β-galactosidase signal
- HEK293T cell line
- DMEM culture medium supplemented with 10% fetal bovine serum
- White 96-well plates
- Transfection of SuperTopFlash and CMV-driven LacZ plasmids
- Lipofectamine 2000 transfection reagent
- Incubation time of 16-18 hours with ligand
- Measurement of luciferase and β-galactosidase signals using a BioTek Synergy 2 plate reader and Dual-Light Reporter System
- Not explicitly mentioned
- Not explicitly mentioned
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