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FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen
- Source :
- Journal of Microbiological Methods. 71:220-224
- Publication Year :
- 2007
- Publisher :
- Elsevier BV, 2007.
-
Abstract
- Salmonella can cause a typhoid fever like disease in genetically susceptible mice. In contrast to in vitro cell culture models, most host cells in infected tissues contain only one or two Salmonella, but a small subpopulation of infected host cells contains many Salmonella. It has been proposed that these various subpopulations have differential relevance during infection but to test this, methods for isolating such Salmonella will be required. We developed a method to purify differentially infected host cells by flow cytometry using Salmonella expressing the green fluorescent protein (GFP). Critical parameters for this method were sufficient GFP expression to detect infected cells against strong host cell autofluorescence, and low variation in GFP content of individual Salmonella. We evaluated more than hundred different GFP-constructs but only one single-copy chromosomal P(sifB)-gfp fusion met these criteria and enabled differentiation of weakly and highly infected cells based on total GFP fluorescence. Confocal microscopy of sorted cells confirmed the successful separation of these subpopulations. In addition to isolation of infected cells, our method also enabled enumeration of the subpopulations and phenotypic characterization by staining with antibodies to surface markers. Surprisingly, a small subpopulation of highly infected host cells contained the majority of Salmonella but based on MHC II and ICAM I expression, this subpopulation was not more strongly activated than weakly infected cells. Our method will facilitate future characterization of the different subpopulations and the identification of bacterial and host factors that control Salmonella load and proliferation in vivo.
- Subjects :
- Microbiology (medical)
Salmonella
Green Fluorescent Proteins
Biology
medicine.disease_cause
Microbiology
Green fluorescent protein
law.invention
Flow cytometry
Mice
In vivo
Confocal microscopy
law
medicine
Animals
Molecular Biology
Salmonella Infections, Animal
medicine.diagnostic_test
Gene Expression Regulation, Bacterial
Flow Cytometry
Phenotype
Molecular biology
Staining
biology.protein
Antibody
Spleen
Subjects
Details
- ISSN :
- 01677012
- Volume :
- 71
- Database :
- OpenAIRE
- Journal :
- Journal of Microbiological Methods
- Accession number :
- edsair.doi.dedup.....fa96a5348f72863e987a98339db7193e