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Keywords:

  • collagenase perfusion;
  • flow cytometry;
  • Kupffer cells isolation;
  • mean fluorescence intensity

Abstract

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.