Immunology and Infection
Published: January 2nd, 2013
A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.
We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.
1. Infection of Mice With P. berghei-ANKA
The results in Fig. 2 show percentages and absolute numbers of different BSL populations recovered from brains of perfused or unperfused malaria-infected and naïve control mice. Isolated BSL were stained with PE-anti-NK1.1 and APC-anti-TCR-β antibodies as indicated in the Protocol text. Consistent with previous findings 7-9, αβTCR+ T cells comprised a high proportion of the BSL pool in brains of perfused malaria-infected mice (day 6 p.i). This population appeared to be significa.......
The isolation and analysis of BSL is a method that allows characterization and quantification of inflammatory cells migrating to the brain in response to tissue injury or infection in experimental mouse models. The introduction of an intracardial perfusion step for removal of the blood compartment before organ extraction and subsequent cell isolation is useful to prevent contamination of inflammatory cells with non-inflammatory circulating leukocytes. This might not be an essential requirement in neurotropic infec.......
The authors would like to thank Miss Liana Mackiewicz for technical assistance. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS and Project Grant 1031212.....
|Name of the reagent
|Solutions and buffers
|Giemsa's azur eosin methylene blue solution
|1:10 dilution in distilled water
|Mouse tonicity PBS
|20 mM Sodium Phosphate, 0.149 NaCl, pH 7.3
|Deoxyribonuclease (DNAse) I
|From bovine pancreas
|30% solution in PBS
|Ammonium Chloride (NH4Cl)
|Red Cell Lysis Buffer
|17 mM Tris,14 nM NH4Cl, pH 7.2
|EDTA disodium salt
|0.1M, pH 7.2
|Antibodies and conjugates
|Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2
|1 μl in 50 μl staining buffer (0.5 mg/50 ml)
|FITC-anti-mouse CD4, clone H129.19
|PE-anti-mouse NK1.1, clone PK136
|PerCPCy5.5-anti-mouse CD8, clone 53-6-7
|APC-anti-mouse TCR-β, clone H57-597
|PE-anti-mouse CXCR3, clone 220803
|Biotinylated-anti-mouse CCR5, clone C34-3448
|Equipment and material
|SuperFrost microscope slide
|Dissection forceps, scissors
|500 ml PBS reservoir
|Cell dissociation kit containing metal sieve
|70 μm nylon cell strainer
|Flow cytometry tubes
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