Ros brite

As previously described, a modified procedure was used to measure the generation of reactive oxygen species (ROS) using flow cytometry (21 (link)). Briefly, at 24 hours post-CLP, peritoneal cells were collected. Following RBC lysis, peritoneal cells were loaded in the dark for 30 minutes at 37°C with 5 μM/mL of ROS Brite™ (10 µM; AAT Bioquest, Sunnyvale, CA, USA), which is a general oxidative stress indicator. As soon as the cells were washed and labeled for neutrophil-specific surface markers, flow cytometry analysis was done.

Cells from peritoneal lavage were plated at 5-10 ×10⁵ cells in 96 well plates and incubated in complete media containing 5 μM/ml ROS Brite™ (10 µM, AAT Bioquest) for 30 minutes at 37 °C. The dye-loading solution was replaced with an HHBS buffer. And then the cells were washed with FACS buffer prior to being stained with the surface markers CD11b and Ly6G to identify neutrophils. FACS analysis was performed using a BD Accuir C6 Plus cell analyzer (BD). All data were analyzed using FlowJo (Tree Star).

Cells were blocked for Fc receptors with anti-mouse CD16/32 antibodies. For surface staining, cells were stained on ice for 30 mins with the following fluorescent conjugated antibodies: fluorescein isothiocyanate (FITC)–anti-Ly6G (1A8, Biolegend), peridinin-Chlorophyll-Protein Complex (PerCP)-anti-Ly6C Abs (HK1.4, Biolegend), phycoerythrin (PE)-anti-CD11b (M1/70, Biolegend) or allophycocyanin (APC)–anti-CD11b (M1/70, Biolegend). Staining for intracellular iNOS with APC–anti-iNOS (CXNFT, ThermoFisher scientific) was performed after fixation and permeabilization of the cells with a Cytofix/cytoperm kit (BD). Neutrophils were identified as CD11b+Ly6G+, and monocytes were identified as CD11b+Ly6G-Ly6C+ (Supplementary Figures 1–4). For in vitro phagocytosis assays, peritoneal cells were incubated with PE-labeled Escherichia coli (Abcam) for 2 hours, then these cells were recollected and labeled with surface markers CD11b, Ly6G, and Ly6C to identify monocytes. For ROS detection, peritoneal cells were incubated in a complete medium containing 5μM/ml ROS Brite™ (AAT Bioquest) for 30 minutes 37℃and then was stained with surface markers CD11b, Ly6G, and Ly6C to identify monocytes. Flow cytometry analysis was performed using the BD Accuir C6 Plus cell analyzer (BD Biosciences).