Hla dr apc cy7 l243

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).

To allow identification of monocytic myeloid cells, PBMCs (5–10 × 106 cells) were stained with PE- Cy7 anti-CD20 (2H7; 560735, BD Biosciences), PE-Cy7 anti-CD3 (SP34-2; 563916, BD Biosciences), APC anti-CD14 (M5E2; 561390, BD Biosciences), BV786 anti-NHP-CD45 (D058-1283; 563861, BD Biosciences), HLA-DR-APC-Cy7 (L243; 307618, BioLegend), BV421 anti-CD192 (CCR2) (48607; 564067, BD Biosciences), FITC anti-CD16 (3G8; 555406, BD Biosciences) and PE-CF594 anti-CD184 (CXCR4) (12G5; 562389, BD Biosciences), as well as Aqua LIVE/DEAD kit (L34966, Invitrogen) to dismiss dead cells. CD45⁺Lin⁻ (CD3 and CD20) was considered as a signature of myeloid cell populations. Monocyte populations were further recognized and sub-categorized according to the expression of CD14 and CD16, where classical monocytes were identified as Lin⁻CD45⁺CD14⁺CD16⁻HLA-DR⁺, intermediate as Lin⁻CD45⁺CD14⁺CD16⁺HLA-DR⁺ and non-classical as Lin⁻CD45⁺CD14⁻CD16-HLA-DR⁺. Data are shown as frequency of CD45 cells or as frequency of HLA-DR cells. Acquisition was then done on an LSRII (BD Biosciences), and marker expression was examined in real-time using the software FACSDiva (BD Biosciences). Data were analysed more in detail with FlowJo version 10.1 (TreeStar, Inc.).