Preparation of Kupffer cell enriched non-parenchymal liver cells with high yield and reduced damage of surface markers by a modified method for flow cytometry.
The aim of this study was to optimise a collagenase perfusion protocol for the isolation of a liver non-parenchymal cell (NPC) suspension enriched for Kupffer cells that reduced damage to F4/80 antigen cell surface expression to allow analysis by flow cytometry. Kupffer cell-enriched liver NPCs were isolated from C57BL/6 mice using different protocols. Flow cytometry was used to examine the effect of collagenase digestion on F4/80 expression on Kupffer cells, and results were represented by the percentage of F4/80 positive cells and by the F4/80 mean fluorescence intensity (MFI). The perfusion temperature, concentration of collagenase solution and total dosage of collagenase for liver perfusion influenced the effect of collagenase perfusion on the expression of F4/80 antigen on Kupffer cells. Collagenase perfusion at 28°C resulted in an increased percentage of F4/80 positive cells (P = 0.001) and MFI (P = 0.005) compared with 37°C. Perfusion with a total dose of 1.0 g/kg BW collagenase (using a 0.75 mg/mL solution) resulted in the highest percentage of F4/80 positive cells (P = 0.001) compared with 0.8 g/kg BW and 1.2 g/kg BW collagenase. Isolation of cells using the modified protocol resulted in a higher percentage of Kupffer cells (P < 0.001) and a higher MFI of F4/80 antigen (P < 0.001) compared with the common protocol.
Liver Research Center, Beijing Friendship Hospital, Capital Medical University, Beijing, PR China.
Fan
F
Xu
Peng
P
Zhen
Yu
Y
Zheng
Feng
F
LIjuan
Yang
Y
Aiting
Cong
C
Min
You
Y
Hong
Jia
J
Jidong
D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002469:Cell Separation; D017364:Collagenases / Q000737:chemistry; D005434:Flow Cytometry; D007728:Kupffer Cells / Q000378:metabolism*; D008099:Liver / Q000166:cytology*; D008297:Male; D008810:Mice, Inbred C57BL; D010477:Perfusion
D000943:Antigens, Differentiation; D015415:Biomarkers; C036763:monocyte-macrophage differentiation antigen; D017364:Collagenases
Kupffer cells isolation; collagenase perfusion; flow cytometry; mean fluorescence intensity