An efficient method to isolate and culture mouse Kupffer cells

• The combination method resulted in a satisfactorily high yield of 5–6 × 106 KCs per liver.
• The results of immunofluorescence and flow cytometry showed that the percentage of F4/80 positive cells accounted for 98.50% and 87.27%, respectively.
• The isolated KCs possess high phagocytic activity.
• The isolated cells had abilities to produce specific cytokines such as TNF-α in response to bacterial endotoxin.

Kupffer cells (KCs) play an essential role in the physiological and pathological functions of the liver. Although the isolation methods of KCs have been well-described, most of them are sophisticated and time-consuming. In addition, these methods are mainly used for isolating the KCs of the human and rat. In this study, a three-step procedure was applied to isolate KCs in sufficient number and purity from mouse liver, including the techniques of enzymatic tissue treatment, gradient centrifugation, and selective adherence. F4/80 immunofluorescence and flow cytometry were used for cell identification. The combination method resulted in a satisfactorily high yield of 5–6 × 106 KCs per liver, over 92.0% positive for F4/80 and 98.5% viable cells. After 24 h of culturing, the KCs showed typical macrophage morphologic features such as irregular shape, transparent cytoplasm and kidney-like nucleus. The phagocytic assay showed that the isolated cells exhibited strong phagocytosis activity. The KCs we isolated were functionally intact and exhibited a concentration dependent TNF-α production induced by LPS. The method we described is an effective method to isolate mouse KCs in high purity and yield, which consuming fewer collagenase and time without altering the functional capacity of the KCs.