Neutrophil Isolation from Peripheral Blood

The preliminary cell isolation—separation of polymorphonuclear cells (PMNs) (contains 91% of neutrophils) from the peripheral blood mononuclear cells (contains 94% of lymphocytes)—was carried out by centrifuging the blood samples in density gradient using Polymorphprep™ reagent (AXIS-SHIELD PoC AS, Oslo, Norway). Briefly, the blood was carefully layered on the reagent, the amount of which was equal to the amount of blood used for separation. Subsequently, the sample was centrifuged at 400 g for 30 min at room temperature. Cells were counted in the Bürker chamber after staining the nuclei with Türk’s solution. To obtain a pure neutrophil fraction (99.9%) from the PMN fraction, an additional isolation step was carried out using magnetic separation (MACS® Separator) with antibodies and magnetic CD16 MicroBeads (catalog no. 130–045-701, Miltenyi Biotec) [42 (link)]. The survival of neutrophils was evaluated using trypan blue under a light microscope, which was found to be 97%.