The use of a flow cytometry-based cytokine analysis for bronchoalveolar lavage with confirmation by real-time RT-PCR in a mouse model of pulmonary inflammation.
Zeidler-Erdely-PC; Erdely-AD; Young-S; Antonini-JM
Toxicologist 2007 Mar; 96(1):103
Bronchoalveolar lavage (BAL) provides a valuable tool for insight into the lung inflammatory state following a toxic or antigenic exposure. Toxicological injury evaluation routinely uses a concentrated first fraction BAL (<600 Ál) to accurately measure various lung inflammatory parameters in mice. However, 100-250 Ál of this BAL fraction is typically used in most conventional assays limiting the researcher to only a few measurements. Recently, flow-cytometry based cytometric bead arrays (CBA) have been developed to simultaneously determine concentrations of multiple analytes in a single 25-50 Ál sample saving both cost and time. In this confirmatory study, we used a mouse inflammation CBA kit to analyze IL-6, IL-10, IL-12p70, MCP-1, IFN-gamma, and TNF-alpha protein in BAL fluid obtained from A/J or C57BL/6J mice exposed to welding fume. Since inflammatory mediators are usually accompanied by both an increase in gene expression and protein, real-time RT-PCR was done on isolated RNA from whole lung homogenates to validate the findings. It was found that a limitation of the CBA kit was sensitivity. In particular, samples with low concentrations of an analyte were often reported as extrapolated below sensitivity or zero thus limiting statistical analysis. These findings made validation by some other method necessary. Real-time RT-PCR strongly supported the findings of the CBA kit by displaying similar trends in the mouse lung inflammatory response caused by welding fume exposure. Therefore, the mouse inflammation CBA kit was an acceptable method for measuring cytokines in studies involving limited sample size and represents a cost efficient screening method for pulmonary inflammation. Also, it is recommended that another method such as RT-PCR or ELISA be performed if increased sensitivity is required.
Laboratory-animals; Animal-studies; Metabolism; Models; Biodynamics; Pulmonary-disorders; Pulmonary-function-tests; Pulmonary-system-disorders; Alveolar-cells; Lung-cells; Lung-irritants; Lung-function
The Toxicologist. Society of Toxicology 46th Annual Meeting and ToxExpo, March 25-29, 2007, Charlotte, North Carolina