Cd62l apc cy7

The following antibody-fluorochrome combinations were used: CD3-PE-Cy7, CD8-Horizon v450, CD4-Horizon v500, IFN-γ-FITC, PD-1-PE, CD45RO-BV650 (all from BD Biosciences, Oxford, UK), CD62L-APC-Cy7 (eBioscience, Hatfield, UK); LAG-3-FITC (R&D Systems, Abingdon, UK), and TIM-3-AF700 (eBioscience, Hatfield, UK). Ex vivo surface staining was performed on 1×10⁶ freshly isolated PBMC. Briefly, one microliter of a 1:50 dilution of each antibody was added to the cells and incubated for 30 minutes, at 4ºC, in the dark. Cells were washed twice with PBS/1% FCS and then resuspended in 200 μL PBS/1% FCS for data acquisition. Flow cytometry data acquisition was performed using a FACSCalibur. Propidium iodide (10 μg/mL) was added immediately prior to acquisition to discriminate dead cells from viable cells. Data analysis was performed using FlowJo (Treestar Inc., San Carlos, CA, version v10).