WNT-3A Conditioned Medium

Mouse L-cells stably expressing WNT-3A, obtained from ATCC, were used to obtain WNT-3A conditioned medium. Conditioned medium obtained from parental L-cells was used as control. Conditioned medium was prepared as per ATCC guidelines. In brief, cells were grown in DMEM (GIBCO, #42430-082) supplemented with 200 units/mL penicillin–streptomycin (GIBCO, #15070-063), 2.5 µg/mL antimycotic (GIBCO, #15290-026), 0.5 mmol sodium pyruvate (GIBCO, #11360070), 10% v/v FBS (Thermo Scientific, #SV30180.03), and 0.4 mg/mL G418 until confluence after which they were split 1:10 and reseeded in uncoated 145/20 mm tissue culture dishes (GBO, #639160) without G418 for 4 days. Medium was collected, centrifuged at 5,000 rpm for 10 min, and passed through a sterile 0.2 µM filter. New medium was added and cells were grown up to day 7, after which medium was collected as before and subsequently mixed with the previous batch.