The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations in...

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.