Anti ki67 pe cy7

Cryopreserved PBMCs were first stained with fixable viability stain 510 (BD Biosciences, San Diego, CA, USA) for 15 min to exclude dead cells, then washed, and surface stained with anti-CD3-BV650 (BD Biosciences), anti-CD4-AF700 (Biolegend, San Diego, CA, USA), anti-CD8-Percp-Cy5.5 (BD Biosciences), anti-CD38-BV786 (BD Biosciences), anti-HLA-DR-Pacific blue (Biolegend), anti-CD160-AF488 (eBioscience San Diego, CA, USA), anti-PD-1-BV605 (Biolegend), anti-TIGIT-APC (eBioscience), and anti-TIM-3-PE (R&D Systems, Inc., USA) at room temperature for 20 min. Cells were permeabilized and fixed with intracellular staining reagents according to the manufacturer’s instructions (eBioscience) before intracellular staining with anti-ki67-PE-Cy7 (Biolegend). Samples were then washed before acquisition and analysis on a BD LSRFortessa flow cytometer instrument with Diva software (BD Biosciences). Relative isotype controls or fluorescence minus one samples were prepared to facilitate gating. Data were analyzed using Flowjo Software version 10 (Tree Star Inc., Ashland, OR, USA).

Cells were stained with the following antibodies (all from eBioscience, except where stated); anti-CD4-BV650 (BioLegend, San Diego, CA, USA), anti-Foxp3-(ef450/FITC), anti-CD11b-AF700 (Biolegend), anti-αv-PE, anti-CD44-APC-Cy7, anti-CD62L-PerCP-Cy5.5, anti-Ki-67-PE-Cy7, anti-KLRG1-ef450, anti-CD45.1-APC, anti-CD51-PE, anti-GM-CSF-PE, anti-IFN-γ-APC, anti-IL-17-PerCPCy5.5. Rat IgG2a and rat IgG1, conjugated to respective fluorophores, were used as isotype control antibodies. For intracellular cytokine staining, cells were re-stimulated ex vivo with 20 µg/ml pOVA overnight, and Brefeldin A added for the last 4 h of incubation. Samples were stained with a fixable viability marker (conjugated with eFluor455, eBioscience) prior to surface staining. For subsequent intracellular antigen staining, samples were washed once in FACS buffer (PBS, 2% FCS, 0.01% NaN₃) and then processed according to manufacturer’s instructions (eBioscience for transcription factors, BD for intracellular cytokines). Flow cytometric data were acquired using a BD LSR Fortessa cell analyzer (BD) and data analyzed using FlowJo software (Treestar version 3.2.1, Ashland, OR, USA).

Thymocytes were first stained with fluorescently labeled antibodies against surface molecules CD4, CD8, CD69, and Qa-2 for 25 min in dark. After extensive washing, cells were fixed and permeabilized using fixation/permeabilization diluent (eBioscience, cat. no. 00-5521) according to the manufacturer’s instructions. Cells were then incubated with anti-p-Stat3-Percp cy5 (BD, San Diego, CA, USA) or anti-Ki67-PE-Cy7 (BioLegend) following the manufacturer’s recommendations.