8 color canto 2 flow cytometer

Fresh whole blood lymphocytes were used for CD4 + T-cell phenotyping without a separation procedure because separation alters chemokine receptor expression. CD4 + T-cell phenotyping has been performed within the 2 h after sampling. We distinguished T-cell subsets according to CD45RA and CCR7 expression: the CM subset (CD45RA -CCR7 + ) and EM subset (CD45RA -CCR7 -) were analyzed. The expression of each HLA-DR, CD40, CD134, CD154, Tbet, and TLR was evaluated in CD4 + T cells and in memory subsets. Leukocytes were labeled with the following antibodies: anti-CD3 V500, anti-CD4 PE-Cy7, anti-CCR7 V450HZ, anti-CD45RA APC or PE-Cy7, anti-CD40 PE-Cy5, anti-CD134 FITC, anti-TLR2 PE (all from BD Biosciences, San Jose, CA, USA), anti-CD4 PerCP-Cy5.5, anti-CD3 APC-eFluor780, and anti-HLA-DR APC-eFluor780 (all from eBioscience, San Diego, CA, USA). RBCs were lysed with Lyse/Fix buffer (BD Biosciences). Cells were fixed and permeabilized using a fix/perm kit (eBioscience) according to manufacturer's instructions. Intracellular markers were detected with anti-CD154 FITC, anti-Tbet AF647 (BD Biosciences), anti-TLR3 FITC, and anti-TLR9 PE (Novus Biologicals, Littleton, CO, USA). Fluorescence was collected on an 8-color Canto II flow cytometer (BD Biosciences).