[Mixed enzyme applied to develop the method on BAlb/c mouse Kupffer cell isolated and cultured in vitro].

ABSTRACT

OBJECTIVE: To research the reliable method for the isolation and culture of Kupffer cell in BALB/c mouse by mixed enzyme. METHODS: Kupffer cells were isolated from liver by in situ perfusion and digestion with 0.1% IV collagenase, 0.2% pronase and 0.01% Dnase I, and by percoll density gradient centrifugation. Kupffer cells were identified by fluorescence microscope, immunohistochemistry and cell endocytosis effect. RESULTS: Kupffer cells were isolated successfully with high purity, the yield of (2-3) 10(6)/per mouse liver and the identification that 0.4% trypan blue indicated that the cells survival rate and purity were more than 96% and more than 92% respectively. The shape of Kupffer cell appeared to be multiplicity, irregularity, polygon and multiangular. Kupffer cells showed lysozyme positive by immunohistochemistry staining. And particles of India ink were found in cytoplasm. CONCLUSION: Here described technique for isolation and culture of Kupffer cells is simple and reliable, and can be used for preparing Kupffer cells with high yield, activity and purity.