Cd14 bb700

Purified monocytes were treated with FcR blocking reagent (Miltenyi) for 10 minutes at 4°C and stained with the following fluorochrome-conjugated antibodies: CD14 BB700 (BD Bioscience), CD40 PE (Miltenyi) and CD86 APC (BD Bioscience) for 20 minutes at RT.
Purified CD8⁺ T cells were stained with the following fluorochrome-conjugated antibodies: anti CD3 BB700 (clone SK7), anti CD8 APCH7 (clone SK1), anti CCR7 BV421 (clone 150503), anti CD45RA- PECy7 (clone H100), anti CD45RO FITC (clone UCHL1), anti CD62L APC (clone DREG-56), anti PD-1 APC (clone MIH4), anti LAG3 PE (clone T47-530), anti-TIM-3 BB700 (clone 344823) for 20 minutes at RT. All antibodies were purchased from BD Bioscience.
All samples were washed after antibody incubation and fluorescence was acquired using the flow cytometer BD FACSVerse. For CD8⁺ T-cells 1x10⁴ events were acquired, while for CD14⁺ cells 5x10⁴. Data were analyzed using BD FACSuit software.

Thawed cells were labeled with viability dye in PBS for 20 min at 37°C, followed by surface staining for 15 min at room temperature in PBS 2% FBS. After stimulation with IFN-α2b 40.000 IU/ml (Miltenyi Biotec) for 15 min at 37°C, cells were fixed and permeabilized using Cytofix Fixation Buffer (BD Biosciences) pre-warmed to 37°C and Perm Buffer III (BD Biosciences) pre-cooled to −20°C, according to the instructions of the manufacturer. Fc Block (Human TruStain FcX, Biolegend) in PBS was used to prevent the unwanted binding of Fc receptor CD16 antibody. Finally, intracellular staining was performed for 30 min at room temperature in PBS with 0.5% BSA. The following panel was used: Viability Dye Aqua Brilliant Violet 510 (L34957 A+B, Invitrogen), CD3 APC-Cy7 (317341, Biolegend), CD19 APC-eFluor780 (47-0199-42, Invitrogen), CD56 APC-eFluor780 (47-0567-42, eBiosciences), CD14 BB700 (566466, BD Biosciences), CD16 Alexa Fluor 647 (557710, BD Biosciences), HLA-DR PE-CF594 (562304, BD Biosciences), and phospho-STAT1 Brilliant Violet 421 (566238, BD Biosciences).