title: [Isolation and purification of primary Kupffer cells from mouse liver].
abstract: Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established.
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Sun
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Qingbo
authors.initials: Q
authors.lastname: Luo
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Xiuxian
authors.initials: X
authors.lastname: Lu
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Daofeng
authors.initials: D
authors.lastname: Zheng
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Diao
authors.initials: D
authors.lastname: He
authors.affiliation: Division of Hepatobiliary Surgery, Department of Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China. *Corresponding author, E-mail: wzjtcy@126.com.
authors.forename: Zhongjun
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology / Q000378:metabolism; D051940:B7-2 Antigen / Q000276:immunology / Q000378:metabolism; D019013:CD40 Antigens / Q000276:immunology / Q000378:metabolism; D015687:Cell Hypoxia; D002469:Cell Separation / Q000379:methods*; D048430:Cell Shape / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D000949:Histocompatibility Antigens Class II / Q000276:immunology / Q000378:metabolism; D007728:Kupffer Cells / Q000276:immunology* / Q000378:metabolism; D008099:Liver / Q000166:cytology*; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D051940:B7-2 Antigen; D019013:CD40 Antigens; D000949:Histocompatibility Antigens Class II; D010100:Oxygen; D014409:Tumor Necrosis Factor-alpha; C067980:CD68 antigen, human; D000097382:CD68 Molecule