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

Fluorescein isothiocyanate-labeled polystyrene microbeads (diameter: 1.0 µm, #17154, Polysciences, Inc., Warrington, PA, USA) were diluted at 1:800 in growth medium, before being added to macrophages that had been seeded in plastic dishes (4 × 10 5 cells/35mm dish for primary PKM, and 1 × 10 6 cells/60-mm dish for IPKM). After being incubated at 37°C, the cells were harvested with TrypLE Express (Thermo Fisher Scientific) at the time points indicated, rinsed with PBS three times to remove non-phagocytosed beads, and fixed with 3.7% formalin in PBS at room temperature for 15 min. After being washed with PBS, the cells were suspended in 0.5 ml of IsoFlow (Beckman Coulter, Fullerton, CA, USA) and then analyzed with a flow cytometer (Epics XL-MCL, Beckman Coulter) to investigate the phagocytosis of FITC-labeled microbeads.