Cell lines and Wnt3a conditioned medium

The mouse monocyte/macrophage-like cell line RAW264.7 (ATCC#TIB-71), the Wnt3a producing cell line, L cells expressing Wnt3a (overexpression mouse Wnt3a ATCC#CRL-2647), and its parent L cell line (ATCC#CRL-2648) were obtained from American Type Culture Collection (USA). The Wnt3a-expressing and its parent L cell lines were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in 5% CO₂. When both Wnt3a and the parent L cell lines were grown to 70−80% confluence, cells were refreshed with DMEM/5% FBS, then the culture media were collected and refreshed at 24, 48, and 72 h. The collected media were pooled and centrifuged at 5000 g for 10 min at 4°C and filtered with 0.22-µm pore filters prior to being aliquoted and stored at -80°C until use. The conditioned media collected from Wnt3a-expressing and parent control L cells were designated as Wnt3a-CM and Ctrl-CM in this study, respectively.