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Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.
- Source :
- Malaria Journal; 2007, Vol. 6, p169-176, 8p
- Publication Year :
- 2007
-
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. [ABSTRACT FROM AUTHOR]
- Subjects :
- MALARIA
LIVER cells
LIVER
CELL separation
CYTOLOGICAL techniques
CELLS
Subjects
Details
- Language :
- English
- ISSN :
- 14752875
- Volume :
- 6
- Database :
- Complementary Index
- Journal :
- Malaria Journal
- Publication Type :
- Academic Journal
- Accession number :
- 34924956
- Full Text :
- https://doi.org/10.1186/1475-2875-6-169