Phenotypic analysis using very small volumes of blood

Analysis of cell-surface phenotype of peripheral blood leukocytes is one of the most common applications of flow cytometry. In mouse research, the small size of the animal limits the amount of blood available. Standard staining methods using lysis of erythrocytes or gradient separation followed by repeated washing involve unavoidable losses of cells that generally limit analysis of blood to terminal methods. Time-course studies, therefore, require sacrifice of groups of mice at each time point. Thus, a method is needed that can be used with much smaller volumes of blood. This will allow serial sampling of the same animal over time, decreasing experimental variability and reducing animal use. The method described here is a no-lyse, no-wash method that uses triggering on a fluorescence parameter. The method allows routine analysis of the phenotype of peripheral blood leukocytes using whole-blood volumes of 20 µl per tube. The data are comparable with values from traditional methods requiring much higher volumes of blood. Due to interference by erythrocytes, light-scatter parameters are not usable with this method. This method has been used for time-course studies of peripheral blood populations in mice lasting as long as four weeks.