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

Macrophages are professional phagocytes belonging to the innate immune system. The phagocytic activity of the IPKM was quantitatively evaluated using flow cytometry. After the addition of FITC-labeled beads to the IPKM culture, the proportion of fluorescence-positive IPKM increased to >95% within 2 h (Figure 3A) . The levels of phagocytosis did not differ significantly between the primary PKM and IPKM (Figure 3A) . To evaluate phagosomal maturation, IPKM were treated with E. coli BioParticles that had been covalently conjugated with pHrodo dye. The intensity of the fluorescence produced by this dye increases if the pH of its surroundings becomes more acidic (18, 19) . Cells exhibiting pHrodo-derived fluorescence appeared after around 30 min incubation, and the number of these cells gradually increased in a time-dependent manner (Video S1 in Supplementary Material). pHrodo-labeled E. coli BioParticles that had been incorporated into IPKM displayed very bright fluorescence after 4 h incubation (Figures 3B,C) , indicating that the phagosomes containing the BioParticles matured into phagolysosomes by fusing with lysosomes, which caused them to become more acidic. These results demonstrate that the IPKM exhibited substantial phagocytic activity.