engleski

rapid flow cytometric method for the detection of neutrophils, monocytes and macrophages in brochoalveolar lavage fluid in mouse model of lung neutrophilia

The aim of this study was to develop a new flow cytometry method for the detection of neutrophils, monocytes and macrophages in the BALF taken from animals used in mouse model of lung neutrophilia. In this animal model, mice challenged intranasaly with LPS respond after 24 hours with influx of neutrophils into the airways. Lung tissue neutrophila is a characteristic pathological hallmark and the neutrophil count in BALF correlate with disease severity1, 2. Differential cell count and determination of neutrophils within BALF cell population are commonly used methods for measurement of inflammatory response in this animal model. Typically, total of 200 – 500 white blood cells (WBC) per slide is counted and cells differentiated based on their morphology. Manual cell count is time consuming and prone to errors due to subjectivity in differentiation and low number of cells counted3. Flow cytometry method has many advantages over traditional method: larger number of cells can be analyzed in shorter time and several parameters can be simultaneously measured: forward scatter (FSC), side scatter (SSC) and up to 12 or more fluorescent parameters4, 5. During the method development we used a wide range of antibodies specific for mouse cell surface markers: CD45, Gr-1, CD49d, CD11b, CD14, CD3, CD 19, F4/80 and CCR-34, 5. Here we present results for the staining procedure for which we found to be most suitable for rapid detection of neutrophils and monocyte/macrophages subpopulation.

cytometry ; neutrophils ; lung neutrophilia mouse model

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