To create stable HEK293 cells expressing the receptor sensor V5-FZD5-FlAsH436-CFP or V5‑FZD5-FlAsH439-CFP, cells were transfected using the Effectene reagent (Qiagen) according to the manufacturer’s instructions. Forty-eight hours after seeding and for a period of 2 weeks, culture medium was replaced every day with medium supplemented with geneticin (G-418) (500 μg/ml) to select for transfected cells. Individual colonies were selected and characterized using confocal microscopy. Positive homogeneous cells expressing the desired constructs were maintained in DMEM-containing G-418 (200 μg/ml).

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