Human fc receptor binding inhibitor purified

For expression analysis of macrophage marker surface proteins and TLR2, undifferentiated and differentiated as well as unstimulated and/or pam3CSK4-stimulated cells were resuspended in Flow Cytometry Staining Buffer (eBioscience, San Diego, CA, USA). To block Fc receptors and prevent nonspecific binding of Fc fragments, cells were incubated with Human Fc Receptor Binding Inhibitor Purified (eBioscience). 1 × 10⁶ cells were stained with anti-TLR2-FITC (mouse monoclonal, FAB2616F, R&D); anti-CD11b-FITC (mouse monoclonal, 11-0113, eBioscience); anti-CD11b-APC (mouse monoclonal, 17-0118, eBioscience); anti-CD14-PerCP-Cy5.5 (mouse monoclonal, 45-0149, eBioscience) or with appropriate isotype controls. Flow cytometric analysis was performed on LSRFortessa (BD Biosciences, San Jose, CA, USA) instrument and analyzed using FlowJo software (Tree Star Inc.). Since these experiments were performed for quality control reasons, to demonstrate the proper molecular characteristics of our cellular models, we do not include these results as figures in the results section. Representative flow cytometry histograms may be found in supplementary material as Supplementary Figure 1 (differentiation markers) and Supplementary Figure 2 (TLR2 expression).

Peripheral blood mononuclear cells were isolated from HC by density gradient separation with Ficoll-Paque PLUS (GE Healthcare). Following erythrocyte lysis with potassium ammonium chloride buffer, monocytes were isolated with CD14 microbeads (Miltenyi Biotec). Monocytes were seeded at a density of 1 × 10⁶ cells/mL in a medium of RPMI 1640 supplemented with 10% FBS, 100 µg/mL L-glutamine (Sigma), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma). They were then stimulated with 300 ng/mL of SLE patient–derived NETs or with an immune complex of NETs plus IgG from SLE patients or HCs (SLE NETs-SLE IgG [10 µg/mL] or SLE NETs-HC IgG [10 µg/mL]) for 2 days at 37 °C in 5% CO₂. Some wells were preincubated for 2 h at 37 °C in 5% CO₂ with 1000 IU/mL IFNα-2b (MSD), as previously reported^{21 (link)}. Following incubation with NETs or an immune complex, monocyte activation was evaluated by the proportion of activated monocytes (7AAD^-CD14⁺CD86⁺HLA-DR⁺) using an 8-color MoFlo XDP (Beckman Coulter). Data were analyzed using FlowJo Software (https://www.flowjo.com/index.php) (Tree star).
Antibodies used for cellular staining were as follows: Human Fc Receptor Binding Inhibitor Purified (eBioscience), CD14-FITC (M5E2, BioLegend), CD86-APC (IT2.2, BioLegend), HLA-DR-APC-Cy7 (L243, BioLegend), and 7-AAD (BioLegend).