Direct isolation of untouched human neutrophils from whole blood in as little as 25 minutes (TECH2P.870)

Abstract Neutrophils are the first line of defense against microbial infections. They sense pathogen-associated molecular patterns through a wide range of receptors including toll-like receptors. Once activated, neutrophils eliminate infection via the phagocytosis of pathogens, releasing proinflammatory cytokines and antimicrobial peptides and producing reactive oxygen species (ROS). The procedures to enrich neutrophils from human whole blood involve at least two steps to remove red blood cells (RBCs) and unwanted leukocytes. For example, neutrophils may be separated from other leukocytes using density gradient centrifugation. However, they are still mixed with RBCs which are then lysed. Alternatively, RBCs are first depleted using lysis or sedimentation followed by fluorescence-activated cell sorting or magnetic cell separation to remove unwanted leukocytes. Here we describe a one-step immunomagnetic, column-free cell separation method which does NOT require RBC lysis, sedimentation or density gradient centrifugation. Non-neutrophils and RBCs are labeled with antibodies and cross-linked to magnetic particles. The labeled cells are removed using an EasySepTM magnet. The complete procedure takes 25-35 minutes. The purity of isolated neutrophils is 96±3% (n=14). The isolated neutrophils are functional and produce ROS upon activation with PMA. This fast isolation method of untouched, quiescent neutrophils facilitates the studies of neutrophil function in inflammatory diseases.