Assessment of flow cytometric tools to characterize milk somatic cells in water buffalo

The aim of this Research Communication was to develop new flow cytometric tools for the fine identification and characterization of milk somatic cells in water buffalo (Bubalus bubalis). Four multicolour panels of antibodies were designed to identify different subsets of live leukocytes and epithelial cells in bulk milk samples. Panel 1, including the CD18/CD172a/CD14/CD16 markers and Live/Dead vitality dye, allowed us to identify total lymphocytes, polymorphonuclear neutrophils (PMN) and monocyte/macrophage subsets. Panel 2 (CD18/CD4/CD8/δ chain/CD335 and Live/Dead dye) allowed us to identify T helper (CD4⁺), T cytotoxic (CD8⁺), γδ lymphocytes and NK cells. Panel 3 (CD18/CD79a/CD21 and Live/Dead dye) allowed us to identify total and CD21⁺ B lymphocytes. Finally, with Panel 4 (CD18/MHC-I/pan Cytokeratin and Live/Dead dye) the epithelial cells were distinguished from leukocytes. In conclusion, we propose a fine characterization of live milk somatic cell (live differential cell count (LDCC)) in buffalo species. In the future the determination of LDCC could used to identify new markers for detecting early inflammatory states of the mammary gland or for monitoring the technological properties of milks of different somatic cell composition.