Isolation and Culture of Human PMNC

Fresh peripheral blood samples were obtained from healthy donors and transferred into plastic Monovette EDTA tubes. The human peripheral mononuclear cells (PMNC) were isolated by Histopaque^® (Sigma-Aldrich, St. Louis, MO, USA), according to the previously described method [58 (link)]. Briefly, 3 mL blood was added carefully over 3 mL of Histopaque^® and centrifuged at 400× g for 30 min at 4 °C. Subsequently, the layer containing lymphocytes and PMNC was transmitted into a new tube and washed several times with PBS. Isolated cells were then maintained in a Panserin 413 medium (PAN-Biotech, Aidenbach, Germany) supplemented with 2% phytohemagglutinin M (PHA-M, Life Technologies, Darmstadt, Germany). Finally, the resazurin assay was performed to assess cell viability as described above.