These authors contributed equally to this work.
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
Article first published online: 24 JAN 2013
© 2013 International Federation for Cell Biology
Cell Biology International
Volume 37, Issue 4, pages 284–291, April 2013
How to Cite
Xu, F., Zhen, P., Zheng, Y., LIjuan, F., Aiting, Y., Min, C., Hong, Y. and Jidong, J. (2013), 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. Cell Biology International, 37: 284–291. doi: 10.1002/cbin.10035
- Issue published online: 11 MAR 2013
- Article first published online: 24 JAN 2013
- Accepted manuscript online: 11 JAN 2013 06:55AM EST
- Manuscript Accepted: 18 SEP 2012
- Manuscript Received: 25 MAY 2012
- collagenase perfusion;
- flow cytometry;
- Kupffer cells isolation;
- mean fluorescence intensity
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.