Anti mouse cd16 cd32 93

Prior to antibody staining, cell suspensions were incubated with purified anti-mouse CD16/CD32 (93, BioLegend, San Diego, CA, USA) to prevent non-specific immunoglobulin binding to Fc receptors. Suspended cells were stained with Zombie Aqua Fixable Viability Kit (BioLegend) to exclude dead cells, incubating at 20℃ for 10 min in advance of antibody staining. To detect cell surface molecules and released NETs, cell pellets were stained with each antibody in PBS containing 2% FBS and 2 mM EDTA for 20 min at 4 °C. Particularly, extracellular CitH3 and PAD4 expressions were detected by performing separate primary (1:500) and secondary antibody (1:500) staining for 20 min at 4 °C [31 (link)]. To detect ROS production, the cells were incubated with 20 µM DCFDA (ab113851; Abcam), a general oxidative stress indicator, for 30 min at 37℃ [18 (link), 32 (link), 33 (link)]. The cells were incubated with a Cell Stimulation Cocktail (plus a Protein Transport Inhibitor) (eBioscience, San Diego, CA, USA) for intracellular staining. The cells were stained with anti-mouse CD4 and permeabilized using a Foxp3/Transcription Factor Fixation/Permeabilization Kit (Thermo Fisher, CA, USA) following the manufacturer’s instructions. Intracellular cytokines were stained with permeabilization buffer (Thermo Fisher, CA, USA).