Generation of Soluble CD155-FLAG Mouse Cells

The sequence of CD155 exons 1–5 was incorporated into the vector p3xFLAG-CMV-13. Consequently, exons 6–8 of the Cd155 gene were replaced with DYKDDDDK to generate mouse sCD155-flag (sCD155). The plasmid generated was transduced into the B16/BL6 mouse melanoma cell line by using Lipofectamine 2000 (Thermo Fisher Scientific), and the cells were selected for resistance to G418 antibiotic (Sigma-Aldrich) and single-cell-sorted by FACS Aria (BD Bioscience). To select one clone with high production of sCD155-FLAG, FLAG-tagged protein in the culture supernatant was detected by using the sandwich ELISA system. A plate was coated with anti-mouse CD155 mAb (TX56, previously generated; Iguchi-Manaka et al., 2008 (link)); the sample supernatant was then applied, followed by biotinylated anti-FLAG (M2) and HRP-conjugated streptavidin. The selected cells were termed “sCD155/BL6.” Empty vector–transduced B16/BL6 was prepared in the same way and was termed “mock/BL6.”