Anti cd14 bv605

PBMCs were stained with the following list of fluorescently labeled antibodies against cell surface markers subsequently detected by flow cytometry: anti-CD14 BV605 (clone: M5E2), anti-CD16 PE-Cy7/BV711 (clone: 3G8), and anti-CD3 PE (clone: HIT3a) from BioLegend; anti-CD20 e450 (clone: 2H7), anti-CD19 e450 (clone: SJ25C1), anti-CD2 e450 (clone: RPA-2.10), and anti-IL-1β (clone: CRM56) from eBioscience; anti-CD56 e450 [clone: B159 (RUO)], anti-HLA-DR APC-Cy7 [clone: G46-6 (RUO)], and anti-CD66b e450 (clone: G10F5) and CCR2 PerCP Cy5.5 from BD Biosciences. Staining protocol proceeded as described above for imaging flow cytometry analysis. Surface Glut-1 receptor expression was detected by binding to the Glut-1 ligand fused to enhanced green fluorescent protein (GFP) (Metafora Biosystems, Evry, France), as previously described (53 (link)). In brief, 10⁶ cells were incubated for 20 min with 10 µl of Glut-1–GFP solution prior to Live/Dead staining using LIVE/DEAD Cell Viability Assay (Life Technologies, CA, USA). GFP fluorescence was detected by staining cells with Alexa Fluor 647 anti-GFP antibody at 1:200 (Biolegend). Data were acquired on a BD Fortessa flow cytometer (BD Biosciences). All compensation and gating analyses were performed using FlowJo 10.5.3 (TreeStar, Ashland, OR, USA).

PBMCs were isolated using Ficoll (GE Healthcare, USA) gradient centrifugation, and were suspended in flow cytometry staining buffer (BD Pharmingen, USA) at a final concentration of 10⁷ cells/ml. After incubation with Fixable Viability Stain, BD Horizon™ Brilliant Stain Buffer, and Fc-blocking antibodies (BD Pharmingen, USA), cells were labeled with antibodies specific for surface markers at 4° C for 30 min. Cells were surface-stained with anti-CD4-FITC, anti-CD3-AF700, anti-CD8-Percp.cy5.5, anti-CD11b-BV510, anti-CD14-BV605, anti-CD16-BV421, anti-CD19-APC, anti-CD45-BV650, anti-CD56-PE (Biolegend, USA). To conduct intracellular cytokine staining, the cells were washed, fixed, permeabilized using the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit, and then stained using anti-IL-10-APC-R7 antibodies (BD Pharmingen, USA). The results were analyzed using FlowJo software version 10.0.7.