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Malar J. 2007 Dec 20;6:169.

Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.

Author information

1
Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2781-901 Oeiras, Portugal. ligdeus@igc.gulbenkian.pt

Abstract

BACKGROUND:

Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity.

RESULTS:

This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1) dissociation reagents choice; (2) cell separation gradient and (3) cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods.

CONCLUSION:

The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.

PMID:
18096071
PMCID:
PMC2244635
DOI:
10.1186/1475-2875-6-169
[Indexed for MEDLINE]
Free PMC Article

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