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4 protocols using cd69 pe

1

Comprehensive Phenotyping of Immune Cells

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The phenotype assay was performed as previously described [25] . In summary, we stained the cell surface for 20 min at 4°C using the following fluorochrome-conjugated antihuman antibodies: CD45RA FITC, CD27 APC, CD3 VioGreen, CD4 PECy7, CD8 APC Cy7 and L/D 7AAD. We employed other antibodies for specific cell populations: CD25 BV421 (BD Horizon, Franklin Lakes, NJ, USA) and CD127 PE-CF594 (BD Horizon) for regulatory T cells (Treg); HLA-DR BV421 (BD Pharmingen, San Diego, CA, USA), CD69 PE (Miltenyi Biotec) and CD25 BV421 (BD Horizon) for activation makers; CD279 (PD1) AF700 (BioLegend, San Diego, CA, USA) and NKG2A BV421 (BD OptiBuild) for exhaustion markers; and, CD103 BV421 (BD Horizon) and CCR7 PE-CF594 (BD Horizon) for chemokine receptor and integrin markers.
The experiments with and without IL-15 incubation O/N and with different concentrations of dexamethasone were performed as described previously. After the staining, cell acquisition was performed using a Navios cytometer (Beckman Coulter), acquiring a mean of 200,000 cells. The analysis was performed using FlowJo 10.7.1 software (FlowJo LLC).
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2

Profiling NK Cell Subsets from Peripheral Blood and Pleural Effusion

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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+CD3CD56+ total lymphocytes.
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3

Splenic T Cell Subsets Quantification

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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).
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Multicolor Flow Cytometry Staining

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The antibodies CD3-BV605 (clone: OKT-3), CD4-PerCP (clone: OKT-4), CD8-PE (clone: RPA-T8) and CD69-PE (clone: FN50) were purchased from Miltenyi (Bergisch Gladbach, Germany). Ficoll was purchased from GE healthcare (Chicago, IL, USA), ultrapure water, DMSO, trifluoroacetic acid (TFA), Tris(2-carboxyethyl)phosphine (TCEP) was purchased form Sigma (MI, USA). All other reagents used were purchased from Thermo Fisher Scientific (MA, USA).
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