Multiparametric Analysis of Neutrophil Biology

FACS: PMNs (5 × 10⁵) were suspended in FACS-buffer (PBS, 10% FBS, 0.1% NaN₃) and stained for 30 min at RT using CD66B-FITC (Biolegend), CD34-PE (Caltag) and CD61-AF647 (Biolegend). Acquisition: Accuri-C6-Plus (Becton Dickinson). PMN (10,000 events) were analyzed using Flow Jo (version 10).
Late apoptosis: isolated neutrophils were incubated in 100 μl annexin binding buffer containing phycoerythrin-conjugated Annexin V (Annexin V-PE) and 7-amino-actinomycin D (7-AAD) (Annexin-V Apoptosis Detection Kit, ThermoFisher Scientific) for 15 min at room temperature (RT) in the dark. After incubation, 400 μl annexin binding buffer was added, and samples were measured immediately on a BD Accuri™ C6 FACS (BD Biosciences). The Annexin V-PE⁺/7-AAD⁺ populations were taken as measurements of late apoptotic cells. Data were analyzed using FlowJo v10 software (FlowJo, LLC).
NETs quantification: PMN (2.5 × 10⁴) were incubated with 0.2 μM SytoxGreen (Life Technologies), 37°C, 5% CO₂, 3 h (13 (link)). PMA (25 nM) was used as the positive control. Results are displayed as mean fluorescence intensity (MFI) measured using a Synergy H1-Hybrid-Reader (Biotek). Excitation: 485 nm/emission: 535 nm.