Cd40 pe

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.

Single-cell suspensions were generated by digestion of confluent cells. A total of 10⁶ cells were incubated with antibodies or an isotype control antibody at 4°C for 30 min in the dark. Samples were washed twice with PBS and analyzed with an FACS Calibur flow cytometer (Becton Dickinson). Specific antibodies used in these analyses were CD34-PE, CD29-PE, CD90-PE, CD45-FITC, CD73-FITC, CD105-FITC, CD14-PE, CD19-PE, CD4-PE, CD8-APC (Biolegend, USA), VEGFR-2-PE, CD144-PE (BD, USA), vWF-FITC (Abcam, USA), CD31-FITC (eBioscience, USA), MHC I-PE, MHC II-PE, CD40-PE, CD80-PE, CD86-PE (Miltenyi Biotec) and isotype control IgG-PE (from Miltenyi Biotec or Biolegend, USA), IgG-FITC (from ebioscience or Biolegend, USA), Flow cytometric data were analyzed using BD CELLQuest software.