Flow Cytometry Staining of Surface and Intracellular Markers

Thawed PBMCs in RPMI 1640 medium were stained with antibodies against CD3-Percp (HIT3a, Biolegend), CD56-BV786 (NCAM, BD Bioscience), CD69-APC/Fire™ 750 (FN50, Biolegend), TIGIT-PE/Cyanine7 (cloneA15153G, Biolegend), CD159a-FITC (REA110, Miltenyi Biotec), CD159c-PE (REA205, Miltenyi Biotec), CD38-Alexa Fluor® 700 (HIT2, Biolegend), and HLA-DR- Brilliant Violet 650™ (L243, Biolegend). NK cells were also stained with the following antibodies including CD3-APC/Cyanine7 (OKT3, Biolegend), CD16- Percp Cyanine5.5 (3G8, Biolegend), CD56-FITC (HCD56, Biolegend), CD96-PE (NK92.39, Biolegend), CD226-APC (11A8, Biolegend), TIGIT-Brilliant Violet 421™ (A15153G, Biolegend); CD155-PE (SKII.4, Biolegend), CD4 (PE/Cyanine7, Biolegend). Another panel of NK cell-specific antibodies, including anti-CD3-APC/Cyanine7 (OKT3, Biolegend), -CD56-BV786 (NCAM, BD Bioscience), and -TIGIT-PE/Dazzle™ 594 (A15153G, Biolegend), were stained for 20 minutes at room temperature. After the PBMCs were washed, fixed and permeabilized with Foxp3/Transcription Factor staining buffer set (Cat: 00-5523-00, eBioscience™) for 40 minutes at 4°C, Ki-67-Brilliant Violet 711™ (Ki-67, Biolegend) were added for staining for 30 min at 4°C. Isotype control mAbs were purchased from the corresponding companies. Cytometer setup and tracking (CST) calibration particles were used to ensure that fluorescence intensity measurements were consistent in all experiments. After the PBMCs were washed with PBS buffer and fixed with 4% formaldehyde solution, the cells were assessed using a BD Fortesa flow cytometer, and dead cells were excluded by staining with LIVE/DEAD fixable viability stain 510. The data were analyzed with FlowJo Software version 10.0 (Treestar, Ashland, OR, USA).