Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

Takenouchi, Takato; Kitani, Hiroshi; Suzuki, Shunichi; Nakai, Michiko; Fuchimoto, Dai-ichiro; Tsukimoto, Mitsutoshi; Shinkai, Hiroki; Sato, Mitsuru; Uenishi, Hirohide

The macrophages were seeded in 8-well chamber slides (Asahi Glass Co., Ltd., Tokyo, Japan) at a density of 2 × 10 5 cells/well. After being washed once with phosphate-buffered saline (PBS), the cells were fixed using 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature. After being washed with PBS containing 0.05% Tween 20 (PBST), the cells were permeabilized with 1% Triton X-100/PBS solution for 10 min and blocked with Blocking One Histo (Nacalai) for 30 min. Then, the cells were incubated with the primary antibodies for 1 h at room temperature in a humidified box. After rinsing the slides with PBST, the EnVision system (DAKO, Hamburg, Germany) was used to visualize antibodyantigen reactions according to the manufacturer's procedure. The immunostained slides were examined under a microscope (Leica, Bensheim, Germany).