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Development of a rapid one-step method using four-color flow cytometry to measure immune cell differentiation and quantification in mouse whole blood.

Young-S; Wolfarth-MG; Porter-DW; Sriram-K; Antonini-JM
Toxicologist 2009 Mar; 108(1):390
The ability to monitor changes in blood cell types is important in understanding the mechanisms and progression of allergy and immune diseases, and further may prove useful in biomarker development. In a mouse model of allergy, a rapid onestep method that uses four-color flow cytometry was developed for immune cell differentiation that requires small amounts of whole blood. Mice (male C57BL/6J) were treated by pharyngeal aspiration on days 1 and 8 to 50 ug ovalbumin or saline (vehicle control). On day 9, whole blood was recovered, and a 100 ul blood was stained with a combination of eight antibodies, incubated for 30 minutes and subsequently lysed. The antibodies consisted of the following: MHCII-FITC, CCR3- PE, CD3-PerCP, CD45R-PerCP, NK1.1-PE, Gr-1-APC, CD4-APC, and CD8a- APC. The procedure allows for identification and quantification of different white blood cells (B lymphocytes, T lymphocytes, CD4+, CD8+, NK, NKT, monocytes, eosinophils, and neutrophils). The Caltag counting beads were added for cell enumeration prior to analysis in FACSCalibur. Samples were acquired through a live gate without compensation. After collecting 3,500 counting beads, the data of all cells were exported to an analysis software, FlowJo. The data were then compensated and analyzed according to the following gating strategy. First, leukocytes were separated by side scattering and forward scattering into three main fractions: lymphocytes, monocytes, and eosinophils + neutrophils. Each fraction was further analyzed using cell-type specific antibodies. A significant (3- to 5-fold) increase in the percentage of monocytes, NK cells, eosinophils, and neutrophils was observed in the circulating blood of ovalbumin-treated mice compared to saline controls. In summary, a rapid four-color flow cytometric method for analyzing immune cell changes in whole blood has been developed for monitoring and assessing cellular responses during immunological and allergic disease states.
Allergic-reactions; Antigens; Biological-effects; Biological-factors; Biomarkers; Blood-analysis; Blood-cells; Cell-differentiation; Cell-function; Cell-metabolism; Cellular-function; Cellular-reactions; Epidemiology; Exposure-assessment; Exposure-levels; Exposure-methods; Immune-reaction; Immune-system; Immune-system-disorders; Immunology; Irritants; Laboratory-animals; Lung-cells; Lung-disorders; Lung-irritants; Microscopic-analysis; Microscopy; Quantitative-analysis; Respiratory-irritants; Respiratory-system-disorders; Statistical-analysis
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The Toxicologist. Society of Toxicology 48th Annual Meeting and ToxExpo, March 15-19, 2009, Baltimore, Maryland