A Novel and Rapid Method to Determine Mast Cell Degranulation in Mixed Peritoneal Cells

Neil Dufton, Nick Goulding, Rod Flower. William Harvey Institute, London, United Kingdom

The suppression of resident mast cell degranulation has long been a therapeutic approach to targeting anaphylaxis and allergic diseases. Current quantitative measurements of mast cell degranulation have been principally restricted to histological analysis, enzymatic markers, such as ß-hexosaminidase, or classical mediators, notably histamine. In recent years some strides have been made to measure changes in light scatter (Perretti et al., 1993) and cell surface markers (Demo et al., 1999) by flow cytometry. This study reports a novel method of identifying degranulation in mixed cell population by characterising the stem-cell factor receptor (C-kit) constitutively expressed by resident mast cells in the context of the transgenic annexin-a1 (ANX-A1) mouse model.

Resident cells were harvested by peritoneal lavage from both BALB/C wild type (WT) and ANX-A1 knockout (KO) mice. The cells were washed and resuspended in a 96 well plate at 1x105 cells/well in PBC buffer (PBS, 1mM CaCl, 0.2% BSA) at 370C. The mixed cells were treated with a concentration range of compound 48/80, a histamine-releasing agent, to induce mast degranulation (Lagunoff et al., 1983) and incubated (370C, 5% CO2) for 10 minutes. The plate was then spun at 2000rpm for 30s and placed on ice, supernatants removed for analysis of histamine release by ELISA and the cell pellets were stained with ckit-PE for flow cytometry analysis. Relative mean fluorescence intensities (MFI) were taken and analysed by one-way ANOVA and post-hoc tests.

A dose dependent increase in side-scatter (SSC), measuring intracellular complexity, and forward-scatter (FSC), indicating cell size, was accompanied by a reduction of C-kit expression by both WT and KO. These responses paralleled a dose dependent release of histamine (Tab.1).

Interestingly mast cells from ANX-A1 KO mice appear more sensitive to degranulation compared to WT. The observed reduction in mast cell specific c-kit expression during degranulation provides a novel technique for assessing this process in both ex vivo and in vivo by flow cytometry.

***P<0.001, **P<0.01, *P<0.05 by One Way ANOVA and Dunnetts post hoc test compared to an un-stimulated control group. ###P<0.001, ##P<0.01, #P<0.05 by one way ANOVA and Bonferroni multi-comparison test compared to the respective WT treatment groups.

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