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J Immunol Methods. 2004 May;288(1-2):111-21.

A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma.

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1
Department of Pulmonary and Critical Care Medicine, Erasmus University Medical Center (Room Ee22-57a), Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. L.vanrijt@erasmusmc.nl

Abstract

Mouse models of allergic asthma are increasingly used to study the immunopathology of this complex disorder. The degree and type of airway inflammation is often studied by determination of differential cell counts on cytospins of bronchoalveolar lavage fluid (BALF) cells stained with May-Grünwald Giemsa, in which the separation of eosinophils (eos) from neutrophils (neutro) and of monocytes (mono) from activated T cells can be quite problematic. In this study, we compared differential cell counts based on morphological criteria on May-Grünwald Giemsa stained cytospins with a newly developed flow cytometric method. BAL fluid cells were identified based on forward and side scatter characteristics (FSC and SSC), autofluorescence of macrophages, and simultaneous one-step staining with antibodies for T cells (CD3-Cy-Chrome), B cells (B220-Cy-Chrome), eosinophils (CCR3-PE), and dendritic cells (DCs) (MHCII-FITC, CD11c-APC). The validity of this flow cytometric determination was tested by morphological analysis of flow-sorted cellular subsets. In an animal model of ovalbumin-induced asthma, this new method correlated very well with the differential counts based on cytospins. Flow cytometric determination of the cellular composition of BAL fluid in mouse models of asthma is a rapid and easy method that can replace differential cell counts based on morphology.

PMID:
15183090
DOI:
10.1016/j.jim.2004.03.004
[Indexed for MEDLINE]
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