Pe conjugated anti cd64

Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated anti-F4/80, purified or PE-conjugated anti-CD21/35 (CR 2/1, clone 7E9) and PE-conjugated anti-I-A/I-E were purchased from BioLegend (San Diego, CA). FITC-conjugated Ly6G (RB6-8C5), PE-conjugated anti-Toll-like receptor 4 (TLR4) and PE-conjugated anti-CD64 were from BD Biosciences (San Jose, CA). U0126 was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany).

Freshly isolated human monocytes and in vitro generated macrophages were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD14, phycoerythrin (PE)-conjugated anti-CD64, anti-CD80, anti-CD206 (BD Biosciences, San Jose, CA, United States) and FITC-conjugated anti-CX3CR1 (BioLegend, San Diego, CA, United States). Monocytes were incubated with antibodies for 30 min at room temperature (RT), then fixed with 4% paraformaldehyde for 15 min at RT, and kept overnight in 4°C prior to analysis. Macrophages were harvested using Macrophage Detachment Solution DXF (PromoCell GmbH, Heidelberg, Germany), stained with antibodies for 30 min at RT and analyzed immediately. Appropriate isotype controls were used as negative controls. Samples were acquired with the BD FACSCanto^TM II system (BD Biosciences). Prior to analysis, monocytes and macrophages were gated based on size forward scatter (FSC) and granularity side scatter (SSC) in order to eliminate other cell types, dead cells, or debris. Analysis was performed using the FlowJo v10.0.7 software (Tree Star, Inc., Ashland, OR, United States). Monocytes were CD14⁺ CD64⁺ CX3CR1⁺ CD206^- CD80^-, whereas macrophages were heterogeneous, with about 50% expressing CD14 and CD64, whereas in general they were positive for CX3CR1 and CD206, and negative for CD80.

The V450-conjugated anti-CD3, Alexa Fluor®700-conjugated anti-CD3, BV650-conjugated anti-CD4, FITC-conjugated anti-CD4, BV605-conjugated anti-CD11b, BV421-conjugated anti-CD11c, BV650-conjugated anti-CD14, PE-Cy^TM7-conjugated anti-CD14, Alexa Fluor®700-conjugated anti-CD16, BV421-conjugated anti-CD16, PE-CF594-conjugated anti-CD16, phycoerythrin (PE)-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD19, Alexa Fluor®700-conjugated anti-CD20, V500-conjugated anti-CD45, BV786-conjugated anti-NKp46, Alexa Fluor®700-conjugated anti-CD56, PE-conjugated anti-CD64, PE-Cy^TM7-conjugated anti-CD86, PerCP-Cy5.5-conjugated anti-CD123, BV711-conjugated anti-CD141, BV786-conjugated anti-PDL2, and PE-CF594-conjugated anti-CD163 antibodies were from BD Biosciences. The APC-Alexa Fluor®750-conjugated anti-HLA-DR antibody was purchased from Beckman Coulter. The PE-conjugated anti-CD1c, APC-conjugated anti-CD303, and APC-conjugated anti-SLAN antibodies were from Miltenyi Biotech. The FITC-conjugated anti-CD192 antibody was purchased from R&D Systems. All antibodies were used according to their manufacturer's recommendations. Antibodies combination for monocytes: CD3, CD11b, CD14, CD16, CD19, CD45, CD56, CD64, CD163, CD192, NKp46, HLA-DR, SLAN, and DC: CD1c, CD3, CD4, CD11b, CD11c, CD14, CD16, CD19, CD20, CD45, CD56, CD86, CD123, CD141, CD303, PDL2, HLA-DR.