Flow-cytometry analysis was performed on 52 frozen peripheral blood mononuclear cells (PBMCs) prior thawing. Egressed cells from one parotid MALT-L and PBMCs were stimulated with PMA (50 ng/mL), ionomycyn (750 ng/mL), Brefeldin-A (10 µg/mL) in RPMI complete (RPMI +10% fetal bovine serum) medium, for 3 hours. Cells were stained using Zombie Aqua Live/Dead kit (Biolegend) for 15 min, washed and incubated for 10 min with human Fc TruStain FcX (Biolegend). Cells were stained for surface antigens, fixed, permeabilised (fixation-permeabilisation buffer; eBioscience) and stained for intracellular cytokines. Antibodies used are listed in online supplementary table S2 . Cells were acquired using a LSR Fortessa II (BD Biosciences) flow cytometer and analysed with FlowJo V.10 software.
Zombie aqua live dead kit
Manufactured by BioLegend
The Zombie Aqua Live/Dead kit is a fluorescent-based cell viability assay. It utilizes a proprietary dye that stains dead cells, allowing for the distinction between live and dead cells in a sample.
Automatically generated - may contain errors
3 protocols using zombie aqua live dead kit
1
Phenotypic analysis of activated PBMCs
2
Murine Blood Cell Flow Cytometry
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Flow-cytometry analysis was performed on mouse blood cells. Blood samples from mice were collected in 1.5 mL Eppendorf tubes with 50 mM EDTA and kept on ice. Red blood cells were removed by incubating the blood samples with 2 mL ammonium chloride-potassium lysing buffer (Thermo Fisher Scientific #A1049201) for 3 to 5 min at RT. Samples were centrifuged at 300 × g for 5 min at room temperature and the supernatant was discarded. This step was repeated once. The cell pellet was then gently resuspended, followed by the addition of 1 mL ice-cold DMEM media and centrifugation at 300 × g for 5 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 500 μL Hanks’ Balanced Salt solution. The cells were stained using Zombie Aqua Live/Dead kit (BioLegend #423101) for 15 min, washed, and incubated for 10 min with human Fc TruStain FcX (BioLegend #101320). The cells were stained for surface antigens. Antibodies used are listed in Table 1 . All samples were acquired using a BD Fortessa and analyzed with FlowJo software (FlowJo, LLC).
3
Comprehensive NK-cell Immunophenotyping of PBMCs
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Flow-cytometry analysis was performed on 43 frozen samples of PBMCs. After thawing, cells were stained with Zombie Aqua Live/Dead kit (BioLegend) for 15 min, washed, and incubated for 10 min with human Fc TruStain FcX (BioLegend). Cells were stained for surface antigens combined in a seven-color panel (CD14, CD3, CD20, CD56, CD16, and NK-receptor). Cells were split in up to five tubes, one for each NK-receptor (NKp46, NKp44, NKp30, NKG2D, and DNAM-1) with 1 million cells per tube. Antibodies used are listed in Supplementary Table S3
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