Expression of killer-cell immunoglobulin-like receptors (KIRs) was determined by flow cytometry. The following antibodies were used: CD158a (clone 143211; R&D systems, Minneapolis, MN), CD158b1/2 (DX27; Biolegend), CD158e1 (DX9; Biolegend), and CD159a (REA110; Miltenyi Biotec). For analysis, CD3− (UCHT1) and CD56+ cells (NCAM16.2; BD Biosciences, San Jose, CA) were gated. KIR phenotype analysis was conducted with WinList, v9.0 (Verity Software House, Topsham, ME). For analysis of expression of natural cytotoxicity receptors and NK-cell G2D, the following antibodies were used: NKp30 (p30-15), NKp44 (P44-8), NKp46 (9E2), and NK-cell G2D (1D11; all Biolegend). HLA status was recognized by W6/32 (Biolegend).
Ncam16
Manufactured by BD
Sourced in United Kingdom
NCAM16.2 is a laboratory equipment product designed for specific research and testing applications. It serves as a tool to perform certain functions within a laboratory setting. Further details about the core function or intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
W6 32, by BioLegend (2 mentions)
Hil 7r m21, by BD (2 mentions)
Anti mouse cd16 cd32 2.4g2, by Cytek Biosciences (2 mentions)
G44 26, by BD (1 mentions)
Fortessa instrument, by BD (1 mentions)
B220 ra3 6b2, by Cytek Biosciences (1 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Gl 183, by Beckman Coulter (1 mentions)
Fes172, by Beckman Coulter (1 mentions)
Human fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Live dead yellow fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Hcd14, by BioLegend (1 mentions)
Rpa t4, by BioLegend (1 mentions)
Fixable live dead stain, by Thermo Fisher Scientific (1 mentions)
Immu510, by Beckman Coulter (1 mentions)
R17 809, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Lh750 haematology analyser, by Beckman Coulter (1 mentions)
Cellquest pro software, by BD (1 mentions)
2331 fun 1, by BD (1 mentions)
8 protocols using ncam16
1
Characterization of KIR and NCR Expression
2
Multicolor Flow Cytometry Immunophenotyping
3
Murine Immune Cell Phenotyping by Flow Cytometry
4
Multiparameter Flow Cytometry of NK Cells
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Fluorescently labeled antibodies to CD56 (NCAM16.2, BD Biosciences), CD3 (SK7 or UCHT1, BD Biosciences), KIR2DL1/2DS1 (EB6B, Beckman Coulter), KIR2DL2/2DL3/2DS2 (GL183, Beckman Coulter or DX27, Biolegend), KIR2DS4 (FES172, Beckman Coulter), KIR2DL4 (mAb33, Biolegend), KIR3DL1 (DX9, BD Biosciences), KIR3DL2 (539304, R&D Systems), and NKG2A (Z199, Beckman Coulter) were used. Dead cells were excluded from the analysis using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). Binding of human IgG to transfectants was determined using PE-labeled anti-human IgG F(ab)2 donkey fragments (Jackson ImmunoResearch).
5
Immune Cell Profiling by Flow Cytometry
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Single-cell suspensions were stained with Live/Dead yellow fluorescent dye (1:1,000, ThermoFisher) for 15 min at room temperature and washed with FACS buffer at 300xg for 5 min at 4oC. Blocking followed with 5 ul human FcR blocking reagent (Miltenyi Biotec) per 100 ul cell suspension for 15 min on ice and staining for blood immune cells was performed with the following anti-human antibodies for 30 min on ice: CD3 (1:54; UCHT1; Biolegend), CD3 (1:27; UCHT1; BD Biosciences), CD4 (1:54; RPA-T4; Biolegend), CD8 (1:54; SK1; Biolegend), CD11c (1:32; 3.9; Biolegend), CD14 (1:68; HCD14; Biolegend), CD14 (1:32; M5E2; BD Biosciences), CD16 (1:68; 3G8; Biolegend), CD19 (1:45; HIB19; BD Biosciences), CD19 (1:32; HIB19; Biolegend), CD33 (1:54; HIM3-4; BD Biosciences), CD45 (1:45; HI30; Biolegend), CD56 (1:27; B159; BD Biosciences), CD56 (1:32; NCAM16.2; BD Biosciences), CD66b (1:54; G10F5; Biolegend), CD123 (1:32; 6H6; Biolegend), CD127 (1:32; HIL-7R-M21; BD Biosciences), HLA-DR (1:68; L243; Biolegend) and Siglec-8 (1:21; 7C9; Biolegend). Cells were centrifuged at 300xg for 5 min at 4oC and resuspended in 1 ml FACS buffer. Data acquisition was performed on a 3 laser-FACS Aria III cell sorter (BD Biosciences) and were analyzed with FlowJo v10 software (BD Biosciences). Sorted neutrophils (40,000) were frozen at -80oC for further processing.
6
Identification of Human and Mouse NK Cells
7
Multiparametric Flow Cytometry Analysis
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For phenotypic analysis of lymphocyte subsets by multi-color flow cytometry, PBMCs were stained with fluorochrome-conjugated antibodies for the surface markers CD3 (OKT3, Biolegend), CD4 (SK3, Biolegend), CD8 (SK1, Biolegend), CD14 (M5E2, Biolegend), CD19 (HIB19, Biolegend), CD56 (NCAM16.2, BD Biosciences), CD94 (DX22, Biolegend), CD161 (HP-3G10, Biolegend), CRACC (235614, R&D Systems) NKG2A (Z199, Beckman Coulter), NKp80 (MA152, Beckman Coulter), and TCRγδ (IMMU510, Beckman Coulter) as well as for intracellular EOMES (WD1928, eBioscience), granulysin (GNLY) (DH2, Biolegend), NKG7 (2G9, Beckman Coulter), and PLZF (R17-809, BD Biosciences). Intracellular HOPX was detected with an unconjugated primary antibody (rabbit polyclonal, Proteintech) followed by a fluorochrome-labeled F(ab)2 anti-rabbit IgG secondary antibody (goat polyclonal, ThermoFisher). Dead cells were identified using a fixable live/dead stain (ThermoFisher).
8
Ex Vivo Immune Profiling of Asthma
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The following antibodies were used for ex vivo phenotyping of peripheral blood obtained from asthma donors: CD3, CD4, CD8, CD11c, CD14, CD16, CD19, CD20, CD56, CD86 and HLA‐DR (SK7, RPA‐T4, RPA‐T8, B‐ly6, MφP9, 3G8, HIB19, 2H7, NCAM16.2, 2331[FUN‐1] and L243, respectively; BD Biosciences, Oxford, UK), ILT3 and Foxp3 (ZM4.1 and PCH101, respectively; eBiosciences, Hatfield, UK) and CD304 (BDCA‐4) (AD5‐17F6; Miltenyi Biotec Bisley, UK). Red blood cells were lysed following staining with BD FACS lysing solution. Foxp3 staining was performed as previously described.18 Samples were subsequently processed on a FACSCalibur flow cytometer running CellQuest Pro software (BD Biosciences), which was also used for analysis. A live dead stain was not used as the flow cytometry staining was performed on whole blood ex vivo.
Absolute and differential blood leucocyte counts were performed routinely using a LH750 haematology analyser (Beckman Coulter, CA, USA) and analysed in conjunction with flow cytometric data to calculate cell numbers.
Absolute and differential blood leucocyte counts were performed routinely using a LH750 haematology analyser (Beckman Coulter, CA, USA) and analysed in conjunction with flow cytometric data to calculate cell numbers.
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