Hbss without calcium and magnesium

Bone marrow cells were isolated from C57BL/6, 129S1, alox5^−/−, and lta₄h^−/− mice (Jackson Laboratories) as described previously with mild modifications (Boxio et al., 2004 (link)). Briefly, mice were euthanized with CO_2, and femurs and tibia were flushed with HBSS without calcium and magnesium (Thermo Fisher Scientific). Spicules or bone matrix were removed by 40 μm cell strainer (Fisher). Red blood cells were lysed in cold NH₄Cl lysis buffer as described previously. (Hurley et al., 2004 (link)) About 1.5 – 2.5 × 10⁷ bone marrow cells were isolated per mouse and 26–40% of the bone marrow cells were CD11b+Ly6G+ neutrophils as confirmed by flow cytometry by using fluorescently-labeled antibodies for CD45, CD11b and Ly6G (Thermo Fisher Scientific). The majority of neutrophils (94%) were morphologically mature and functionally competent, as reported previously. (Boxio et al., 2004 (link)) This technique allows for rapid isolation of 17-fold more neutrophils than those isolated from peripheral blood per mouse. Additional neutrophil purification was performed using the EasySep mouse neutrophil enrichment kit (STEMCELL), following the manufacturer’s recommended protocol. The purity of CD11b+Ly6G+ neutrophils was 65-75% as evaluated by flow cytometry analysis.

BMDMs were plated in half-area black 96-well plates in RPMI without phenol red (Invitrogen). Cells were incubated for 10 min at 37°C with 5 μM MitoSOX (Thermofisher). Stimuli were added and fluorescence (Ex₅₁₀, Em₅₈₀) recorded using a Synergy plate reader (BioTek, Winooski, VT). Neutrophils in HBSS without calcium and magnesium (ThermoFisher) were incubated for 1 h with 100 nM PMA (Enzo Life Sciences, Farmingdale, NY) and 5 μM MitoSOX during the last 10 min of incubation. ROS were measured by flow cytometry.