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5 protocols using cd69 bv510

1

Comprehensive Immune Cell Phenotyping

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To analyze cell surface activation markers, PBMCs were stimulated by 1 μM TLR7/8 agonists or DMSO (as a negative control) for 24 h at 37°C. Then cells were collected and washed in FACS buffer (PBS + 2%FBS). Cells were incubated by fluorescence antibodies at 4°C for 30 min. For T cells, CD3-PE-Cy7 (Biolegend), CD4-PE (BD), CD8-FITC (Biolegend), CD25-BV421 (Biolegend), CD69-BV510 (Biolegend). For NK cells, CD16-PE (Biolegend), CD56-APC (Biolegend), CD69-BV510 (Biolegend), NKG2D-BV421 (Biolegend). For monocytes, CD14-PE-Cy7 (Invitrogen), CD40-APC-Cy7 (Biolegend) HLR-DR-BV510 (Biolegend). For dendritic cells, anti-human lineage cocktail-APC (Biolegend), CD11c-PE-Cy7 (Biolegend), CD80-BV510 (Biolegend), CD86-AF488 (Biolegend).
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2

T Cell Activation Profiling Protocol

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T cell activation with CD3/CD28 activating beads was controlled with CD25 and CD69 activation markers. Approximately 150,000 were fixed in 3% PFA for 10 min at RT. Cells were washed in 1% FBS/PBS and stained with the corresponding antibody for 45 min on ice, (1:50 dilution was used for CD25 FITC, #555431 from BD and CD69 BV510 #310929 from Biolegend). Cells were extensively washed and profiled using BD FACSVerse™ instrument. Gates for activation marker-positive cells were set by utilizing unstained controls. FlowJo software was used for the data analysis. Gating strategy is described in Supplementary Fig. 7.
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3

Quantifying NK Cell Markers in Whole Blood and Tumors

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Whole blood and intratumoural immune cells were stained with CD56-FITC-Viobright, NKG2A-APC, CCR5-FITC, CCR1-APC, CCR3-PE (Miltenyi Biotec), CD3-APC-Cy7, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510 (BioLegend). Red blood cells were lysed using BD Lysing Solution (BD Biosciences) as per manufacturer’s instructions. NK cells were quantified as CD56+CD3 cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (Tree Star).
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4

Evaluating NK Cell Response to Olaparib

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Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by density gradient centrifugation and resuspended in RPMI supplemented with 10% FBS and 1% penicillin–streptomycin (complete RPMI (cRPMI), Gibco). PBMC were seeded at a density of 500,000 cells per 500 µL of cRPMI and treated for 24 h with 5 μM or 10 μM of Olaparib. Following treatment, the cells were surface stained for flow cytometric analysis using CD56-FITC (BioLegend), CD3-APC-Cyanine7 (BioLegend), and the following antibodies for activating NKRs, NKp46-PE-Cyanine7 (BioLegend), DNAM-1-PE (BioLegend), NKp30-BV421 (BioLegend), NKG2D-PE-Cyanine5 (BioLegend), CD16-PE-Cyanine7 (BioLegend), inhibitory receptors NKG2A-APC (Miltenyi Biotec), PD-1-PE-Cyanine7 (BioLegend), and TIGIT-PerCP-Cyanine5.5 (BioLegend), death receptor ligands TRAIL-APC (BioLegend) and FasL-BV421 (BioLegend), and phenotypic markers, CD57-PE (BioLegend), CD69-BV510 (BioLegend), and CD27-Pacific blue (BioLegend). All samples were acquired using the BD FACS CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo v10 software (BD Biosciences). NK cells were defined as CD56+ CD3 in the lymphocyte gate. The gating strategies are available in Supplementary Figure S1.
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5

NK Cell Profiling in Obese OAC

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PBMC were isolated from non-cancer controls by density gradient centrifugation and seeded at a density of 1 × 106 cells/ml RPMI supplemented with 10% FBS and 1% pen/strep. Cells were treated with ACM or TCM from non-obese or obese OAC patients for 2 or 24 h. Cells were stained with CD56-FITC-Viobright, NKG2A-APC (Miltenyi Biotec), CD3-APC-Cy7, CD71-PE-Cy7, CD36-PerCP-Cy5.5, NKp30-BV421, NKp46-PE-Cy7, NKG2D-PE-Cy5, PD-1-PE-Cy7, TIGIT-PE-Cy5, CD69-BV510, TRAIL-APC and FasL-BV421 (BioLegend). NK cells were quantified as CD56+CD3 cells within the lymphocyte gate. Cells were acquired using the CANTO II (BD Biosciences) flow cytometer and analysed using FlowJo software (BD Biosciences).
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