Neutrophil Isolation from Whole Blood

20 mL of the whole blood on lithium heparin anticoagulant was collected from sepsis, NMOSD, periodontitis patients and HC. Neutrophils were purified by negative selection by microbeads, which allowed the removal of DCs, B cells, monocytes, macrophages, activated T cells, and activated NK cells (MACSxpress Whole Blood Neutrophil Isolation Kit, Miltenyi Biotec GmbH, Germany). Residual erythrocytes were lysed using 2mL ammonium chloride Lysing Reagent (BD Biosciences) for 5 min. The final purity of the neutrophil population was assessed by flow cytometry using CD14-PE (clone M5E2), CD15-FITC (MMA), and CD16-PECy7 (3G8, all from BD Pharmingen) mAbs. Flow cytometric analysis of the isolated cell population showed that the percentage of CD15^highCD16⁺CD14⁻ neutrophils was > 98%. The level of contaminating CD14⁺CD15⁺ monocytes was about 0.4%, and CD15⁺CD16⁻ eosinophils was < 0.1% after isolation, as we proved in our previous studies [7 (link), 9 (link), 19 (link)].