Flow Cytometric Analysis of Xenopus Immune Cells

Flow cytometry is a versatile analytical platform capable of multiparameter analysis of more than a thousand individual cells per second. This technique is used to measure the physical and chemical characteristics of individual cells in a heterogeneous cell suspension as they pass through one or multiple lasers. Physical properties, such as size and internal complexity, are recorded as light scattering at different angles and are expressed as forward- and side-scatter, respectively. Following light excitation, fluorochromes conjugated to antibodies or intercalated with different cellular components reemit light at distinct wavelengths. This can identify a broad array of cell specific antigens, further defining distinct cell subsets based on activation, lineage, and developmental stage. The combination of labels that can be used depends on the laser used to excite the fluorochromes and on the detector and available antibodies. With the growing number of Xenopus-specific antibodies, flow cytometry can be used to identify, isolate, and characterize distinct immune cell subsets. In this protocol, different methods to obtain single-cell suspensions from various X. laevis tissues are described. A standard three-parameter procedure defining viability and two cell-surface markers is then described.