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2 protocols using nkp30 pe

1

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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2

Comprehensive Immune Cell Profiling

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Antibodies (CD8 Vioblue, CD3 Viogreen, vγ9 vδ2 FITC, CD4 PE, CD45 Per-CP, vγ9 vδ1 APC, CD56 APC-Vio770, CD16 Vioblue, NKG2D PE, PD-1 Per-CP, CD158b PE-Vio770, NKG2A APC, TCR-γδ APC-Vio770, NKp30 PE) (Miltenyi Biotec, San Diego, CA) were added to 25 µL of whole blood at the proper titrated volumes and allowed to incubate for 30 minutes at room temperature in the dark. Blood samples were lysed with 1X Red Blood Cell Lysis solution (Miltenyi Biotec, San Diego, CA), washed twice in phosphate-buffered saline (PBS), and resuspended in 200 µL PBS.
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