Ficoll paque plus separation medium

Anticoagulant citrate dextrose solution-treated fresh blood was layered on Ficoll-Paque Plus separation medium (GE Healthcare Life Science) at room temperature for 45 minutes to allow erythrocytes to sediment. Leukocytes with minimal residue erythrocytes after sedimentation were carefully layered on the top of 10 ml Ficoll-Paque Plus separation medium and centrifuged at 400g for 40 minutes without brake at room temperature. Neutrophils and peripheral blood mononuclear cells (PBMC) were then recovered to separate tubes [49 ].
Leukocytes (approximately 1x10⁶) were stained with fluorescent conjugated antibodies for CD16 (3G8), CD66b (G10F5) from Biolegend, CD177 (MEM-166) from Abcam and CD177 (REA258) from Miltenyl Biotec in Ca⁺⁺Mg⁺⁺ free HBSS. Data were acquired on a FACSCanto II flow cytometer (BD Bioscience) and analysed using FlowJo software (TriStar). 1–2 million of CD177^neg and CD177^hi subsets were sorted on a BD FACS Aria II from CD66b⁺ neutrophils for sequencing and RNA analysis.

Blood samples (20 ml) were drawn from 23 healthy individuals after obtaining written informed consent. Subjects who were affected by health conditions, including allergic or infectious diseases, or who took medication for such conditions were excluded from the study. To isolate peripheral blood mononuclear cells (PBMCs), each blood sample was diluted with an equal amount of phosphate-buffered saline (PBS; Gibco BRL/Invitrogen Technologies, Carlsbad, CA, USA) and overlaid onto Ficoll-Paque PLUS separation medium (GE Healthcare, Buckinghamshire, England). The cells at the plasma/Ficoll interface were collected and washed with PBS containing 10 mM EDTA and 2% fetal bovine serum (FBS), followed by a wash with RPMI 1640 medium containing 10% FBS. The total number of PBMCs was counted using a C-chip cell counter (NanoEnTek, Seoul, Korea). The cells were then cryopreserved at a concentration of approximately 1×10⁷ cells/mL in RPMI 1640 medium containing 10% FBS and 10% dimethyl sulfoxide (Wako, Osaka, Japan). Immediately after isolation, the cells were frozen at −80°C for 24 h. Subsequently, the cells were kept in liquid nitrogen until they were subjected to cell sorting. All experimental procedures described in this article were carried out according to a protocol approved by the Ethics Committee of Tohoku University Graduate School of Medicine.