Apc conjugated anti human il 10

The percentage of CD4⁺CD69⁺IFNγ⁺, CD8⁺CD69⁺IFNγ⁺, CD4⁺CD69⁺IL-17⁺, CD4⁺CD69⁺IL-10⁺ and CD8⁺CD69⁺IL-10⁺ in individual samples was determined by flow cytometry following intracellular staining with anti-cytokine monoclonal antibodies. Upon venipuncture, whole peripheral blood was incubated with Ionomycin (Io) and Phorbol Myristate Acetate (PMA) for 4 h. Following activation, whole peripheral blood was stained with FITC-conjugated anti-human CD8, PE-conjugated anti-human CD69, and PerCP-conjugated anti-human CD3 for 30 min; then fixed and permeabilised using BD FACS^TM Permeabilizing Solution (BD Biosciences), followed by intracellular staining with APC-conjugated anti-human IL-10, APC-conjugated anti-human IL-17A and APC-conjugated anti-human IFNγ. All monoclonal antibodies were supplied by Becton Dickinson (San Jose, CA, United States). The FACS CANTO II cytometer (Becton Dickinson, San Jose, CA, USA) was used to acquire at least 20000 with data analysed on BD FACSDiva™ 6.0 Software. Figure 1 shows the gating strategy to quantify the intracellular cytokine production (IFNγ, IL-17 and IL-10) on peripheral CD4⁺ and CD8⁺ T lymphocytes.