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).
Nkp46 pe cy7
Manufactured by BioLegend
NKp46-PE-Cy7 is a fluorescently-labeled antibody that targets the NKp46 receptor expressed on natural killer cells. It is designed for flow cytometry applications to identify and quantify natural killer cells.
Automatically generated - may contain errors
Lab products found in correlation
Canto 2, by BD (2 mentions)
Pd1 pe cy7, by BioLegend (2 mentions)
Cd69 bv510, by BioLegend (2 mentions)
Cd56 fitc viobright, by Miltenyi Biotec (2 mentions)
Nkg2a apc, by Miltenyi Biotec (2 mentions)
Nkp30 bv421, by BioLegend (2 mentions)
Nkg2d pe cy5, by BioLegend (2 mentions)
Tigit pe cy5, by BioLegend (2 mentions)
Cd3 fitc, by BioLegend (2 mentions)
Cd3 apc cy7, by BioLegend (1 mentions)
Lysing solution, by BD (1 mentions)
Ccr5 fitc, by Miltenyi Biotec (1 mentions)
Ccr1 apc, by Miltenyi Biotec (1 mentions)
Ccr3 pe, by Miltenyi Biotec (1 mentions)
Foxp3 pe, by BioLegend (1 mentions)
Cd25 apc cy7, by BioLegend (1 mentions)
Cd45 percp, by BioLegend (1 mentions)
Cd11b v500, by BioLegend (1 mentions)
Cd19 bv786, by BioLegend (1 mentions)
Cd8α bv650, by BioLegend (1 mentions)
6 protocols using nkp46 pe cy7
1
Quantifying NK Cell Markers in Whole Blood and Tumors
2
Neonatal Lung Immune Cell Profiling
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Neonatal lung single‐cell suspensions were used for all immunostaining. Panels of monoclonal antibodies (purchased from BD Bioscience unless otherwise stated) were developed to enable phenotypic characterisation of leucocytes of myeloid: CD45‐PerCP (clone 30‐F11), CD11b‐v500 (clone M1/70), CD11c‐AF700 (clone HL3), CD19‐BV786 (clone 1D3), CD103‐PE (clone M290), CD301‐PE‐Cy7 (clone MGL1/MGL2; BioLegend), F4/80‐FITC (clone BM8; BioLegend, San Diego), Ly6G/C‐APC‐Cy7 (clone RB6‐8C5), I‐A/I‐E‐AF647 (clone M5/114.14.2) and B220/CD45R‐PE‐CF594 (clone RA3‐6B2) and lymphoid: CD45‐PerCP (clone 30‐F11), NKp46‐PE‐Cy7 (clone 29A1.4; BioLegend), CD19‐BV786 (clone 1D3), CD3‐FITC (clone 17A2), CD4‐v500 (clone RM4‐5), CD8α‐BV650 (clone 53‐6.7), CD25‐APC‐Cy7 (clone PC61) and Foxp3‐PE (clone FJK‐16s) lineages. Intracellular staining for Foxp3 was performed using an intracellular Foxp3/Transcription Factor Staining Buffer Kit (eBioscience, San Diego). All samples were kept as individuals. Immune cell phenotypic characterisation was performed using the FlowJo software (version 10.6.1; BD Bioscience). Fluorescent minus one staining controls were used for all panels where necessary. Flow cytometry data quality was based on primary time gates to ensure appropriate laser delay (pre‐determined by automated CS&T) during sample acquisition.
3
Phenotypic Analysis of Ex Vivo Activated NK Cells
4
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).
5
NK Cell-Mediated RVFV Gn Protein Immunity
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Three micrograms per milliliter RVFV Gn protein (custom; GenScript)-coated MaxiSorp plates (Thermo Fisher) were blocked with 5% BSA in PBST (0.01%) for 1 h at 37°C. MAbs were added to wells at 5 μg/ml and incubated for 2 h at 37°C. NK cells were isolated by negative selection from C57BL/6 mouse spleens using EasySep mouse NK cell isolation kit (StemCell Technologies). Purified NK cells were added at 2 × 105 cells/well in the presence of brefeldin A (Sigma-Aldrich), GolgiStop (BD), and anti-CD107a conjugated to phycoerythrin (PE) (BioLegend clone 1D4B) to wells already containing Gn/MAb. NK cells were incubated for 5 h at 37°C. Cells were then washed and stained with near-infrared (IR) fluorescent reactive dye (Thermo Fisher). Cells were stained for cell surface markers CD3 allophycocyanin (APC)-Cy7 (BioLegend clone 17A2), CD11b fluorescein isothiocyanate (FITC) (BioLegend clone M1/70), and NK1.1 APC (BioLegend clone PK136). The purity of NK cells was confirmed by CD3 APC (BioLegend clone 17A2), CD19 BV421 (BioLegend clone 6D5), NKp46 PE-Cy7 (Biolegend clone 29A1.4), and CD14 APC-Cy7 (BioLegend clone Sa14-2) staining. All cells were fixed in BD Cytofix/Cytoperm and then analyzed by flow cytometry on a BD LSRFortessa flow cytometer. All flow cytometric data were analyzed using FlowJo 10.7.1.
6
NK Cell Surface Marker Profiling
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NK3.3-LTV cells were analyzed for changes in cell surface marker expression compared to non-immortalized NK3.3 cells. All cells (1 x 105 cells per antibody) were centrifuged at 300 g x 5 min, media aspirated, and 1.5 µL single antibody + 20 µL staining buffer (phosphate buffered saline (PBS) + 1% bovine serum albumin + 1% sodium azide) were added to cells. Sample tubes were incubated for 30 min on ice, followed by addition of 200 µL staining buffer and incubated for another 10 min on ice. Samples were analyzed using LSRFortessa Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using FlowJo software. Antibodies used include: NKp46 PE/Cy7 (#331915, BioLegend, San Diego, CA); NKp30 PE (#325207, BioLegend); NKp44 PE (#IM3710, Beckman Coulter, Brea, CA); CD94 PE (#130-098-974, Miltenyi Biotec, Auburn, CA); NKG2D APC (#120-003-706, Miltenyi Biotec); CD161 APC-Alexa Fluor750 (#B30630, Beckman Coulter); CD158e1 BV421 (#312713, BioLegend); CD158a,h PE/Cy7 (#A66899, Beckman Coulter); CD244 PerCp Cy5.5 (#B21171, Beckman Coulter); CD16 ECD (#A33098, Beckman Coulter); CD133 APC (#130-098-829, Miltenyi Biotec); CD57 BV421 (#563896, BD Biosciences); CD158 FITC (#339503, BioLegend).
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