Cd69 pe

Total mononuclear cell suspension derived from peripheral blood and pleural effusion samples was obtained by ficoll hystopaque (Lonza, Basel, Switzerland) gradient stratification [25 (link)]. To identify NK cell subsets, cells obtained were subsequently stained with monoclonal antibodies against surface markers (CD14-PE and CD45-FITC, CD3-PerCP, CD56-APC, CD16-FITC, CD9-PE, CD49a-PE, CD57-PE, CD69-PE, NKp30-PE, NKG2D-PE, and NKG2A-PE) all purchased from Miltenyi Biotec (Auburn, CA) and analysed by a Becton Dickinson (BD) FACSCanto II flow cytometer (Becton Dickinson, CA). Briefly, after physical parameter analysis (FSC/SSC), CD45⁺CD14⁻ lymphocytes were gated and assessed for NK markers. NK cells were gated on CD45⁺CD3⁻CD56⁺ total lymphocytes.

The splenocytes were transferred to RPMI 1640 medium for cell culture to reach a density of 1×10⁷ cells/mL. To measure the percentage of naive T cell and activated T cell, a total of 200 μL cell suspension of each specimen was incubated at 4 °C in the dark for 30 min after adding CD4-FITC (eBioscience, CA, USA) and CD62-PE (Miltenyi Biotec, NRW, Germany), CD4-FITC and CD69-PE (Miltenyi Biotec) antibodies, respectively. Then the supernatant was removed and cells were resuspended using 1× PBS. To detect the Treg cells proportion, the splenocytes were stained with CD4-FITC and CD25-APC (eBioscience) for 20 min. Then the cells were fixed, permeabilized and stained with labelled Foxp3-PE antibody (eBioscience). To evaluate the percentage of Th17 cells, splenocytes were stimulated with a cell stimulation kit (BD Pharmingen, CA, USA) for 6 h. Cells were stained with CD4-FITC for 20 min at room temperature, then followed by a working fixation solution and permeabilization buffer. Intracellular staining was incubated with IL-17-PE antibody (eBioscience) for 20 min at room temperature. The flow cytometry analysis was performed using a CytoFLEX flow cytometery (Beckman Coulter, Inc., CA, USA).