Neutrophil Metabolic and Functional Assays

Neutrophils (0.3 × 10⁶) were activated with Zy/op (100 µg/mL) for 1 h, at 37 °C and 5% CO₂. Neutrophils were washed in PBS and incubated with MitoSOX (5 µM, Thermo, M36008) or Mitotracker Red (50 nM, Thermo, M7512) and Green (100 nM, Thermo, M7514) for 30 min. Samples were washed twice in PBS and immediately analysed by flow cytometry.
For glucose uptake assay, neutrophils (0.5 × 10⁶) in glucose-free RPMI medium supplemented with the fluorescent glucose analogue 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG, Invitrogen, 30 µM, N13195) were activated with Zy/op (100 µg/mL) for 30 min, at 37 °C and 5% CO₂. Neutrophils were washed twice in PBS and immediately analysed by flow cytometry.
To evaluate the expression of surface antigens, neutrophils were incubated with specific antibodies to GLUT1 (1:200, Abcam, Cat# ab209449), Ly6G (1:200, BD Bioscience, Cat# 560599) CD15 (1:100, BD, Cat# 562370) or CD11b (1:200, Biolegend, Cat# 101212) or the appropriate isotype controls for 1 h. Viable cells were assessed by incubating cells with Fixable Viability Dye (Thermo).
The fluorescence of samples was measured in a flow cytometer (FACSVerse™, BD Biosciences) and analysed using FlowJo software (Tree Star). Ten thousand cells were analysed^{52 (link)}.