Vivid pacific blue

Fixed, cryopreserved cells were thawed, permeabilized and stained for cellular markers: T-lymphocyte markers (CD3 and CD8), and intracellular cytokine markers (Th1/Tc1: IFN-γ and IL-2; Th2/Tc2: IL-13; Th17/Tc17: IL-17; and the proliferation marker: Ki67). Expression was measured by multiparameter flow cytometry (BD LSR Fortessa) using the following monoclonal antibodies: anti-CD3-APC-Cy7 clone UCHT1, anti-IFN-γ-Alexa Flour 700 clone B27 and anti-IL-17-PE-Cy7 clone BL168 (Biolegend, San Diego, California, USA); anti-CD8-PerCP-Cy5.5 clone SK1, anti-Ki67-FITC clone B56, anti-IL-2-APC clone 5344.111, anti-IL-13-PE clone JES10–5A2 (BD Biosciences, San Jose, California, USA); VIVID-Pacific Blue (Invitrogen, Brown Deer, Wisconsin, USA). Samples were acquired on a BD LSR Fortessa flow cytometer.

Cells were thawed, permeabilized and stained for viability (VIVID), proliferation, using Ki67[18 (link)], and the following intracellular cytokines with optimized concentrations of fluorescence-conjugated antibodies: anti-CD3-allophycocyanin (APC)-cyanine 7 (Cy7; UCHT1) (Biolegend), anti-CD8-peridinin chlorophyll protein-Cy5.5 (Sk1) (BD), anti-Ki67-fluorescein isothiocyanate (B56) (BD), anti-IFN-γ-Alexa Fluor 700 (B27) (Biolegend), anti-IL-2-APC (5344.111) (BD), anti-IL-13-pycoerythrin (JES10-5A2) (BD), anti-IL-17-phycoerythrin-Cy7 (BL168) (Biolegend), and VIVID-Pacific Blue (Invitrogen). Samples were acquired on a BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA).