The phenotype of eNK cells was analyzed weekly using flow cytometry. The following mouse anti-human antibodies were used for staining: CD16 (3G8), CD56 (NCAM 16.2), NKG2D (1D11), DNAM-1 (DX11), CD3 (HIT3a) from BD PharMingen; CXCR1 (8F1), CXCR3 (CEW33D), CXCR4 (L276F12), CXCR6 (K041E5), CCR4 (L291H4), CCR5 (J418F1), CCR6 (G034E3), CCR9 (L053E8), CX3CR1 (2A9-1), Fas-L (NOK-1), IFNγ (B27) from BioLegend; CD62L (DREG-56), intracellular Perforin (dG9), Granzyme B (GB11), and TNF-α (MAb11) from eBioscience. For surface staining, NK cells were resuspended in FACS buffer (0.5% FBS and 2 mM EDTA in PBS), blocked with 10% mouse serum for 30 min, then incubated with the indicated antibodies for 30 min at 4°C using concentrations recommended by the manufacturers. For subsequent intracellular staining, NK cells were stained using the BD Cytofix/Cytoperm Kit following manufacturer protocol. Data was acquired using a BD FACS Accuri C6 and analyzed using FlowJo v10.2 software. Isotype IgG antibodies were used as negative controls. Human NK cells were defined as CD16+, CD56+ and CD3−. Mouse anti-human HLA-ABC (W6/32), MICA/B (6D4), CD155 (2H7) and PD-L1 (MIH1) from eBioscience, ULBP-1 (170818) from R&D, and Fas (DX2) from BioLegend were used to determine MHC-I, NKG2D ligand and DNAM-1 ligand expression on TC106 with surface staining performed as above for NK cells.
Ulbp 1 170818
Manufactured by R&D Systems
ULBP-1 (170818) is a laboratory instrument designed for measuring biomolecular interactions. It utilizes label-free optical biosensing technology to detect and analyze the binding of analytes to immobilized ligands. The core function of this product is to provide researchers with a tool for real-time monitoring and quantification of molecular interactions.
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Lab products found in correlation
Tnfα mab11, by Thermo Fisher Scientific (1 mentions)
Cd62l dreg 56, by Thermo Fisher Scientific (1 mentions)
Dnam 1, by BioLegend (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Rpa 2, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
2 protocols using ulbp 1 170818
1
Phenotyping of Expanded Natural Killer Cells
2
Comprehensive Immunophenotyping of Hematologic Malignancies
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Monoclonal antibodies (mAbs) were used to define blast populations as follows: AML: CD45 (BD, Hl30), CD34 (BD, 581/CD34), CD117 (Invitrogen,104D2), HLA-DR (BD, G46-6) and CD33 (Biolegend, WM53). T-ALL: CD45, CD5 (Biolegend, L17F12), CD7 (BD, M-T701), CD2 (BD, RPA-2.10), CD3 (BD, SK7) and CD8 (BD, RPA-T8). The following mAbs were used to define T cell subsets: CD3(BD, UCHT1), CD4 (BD, RPA-T4), CD8 (BD, RPA-T8)), CD45RO (BD, UCHL1), CD62L (BD, DREG-56), CD95 (Biolegend, DX2). RhNKG2D Fc chimera (R&D, 12990NK-050) and rhIgG1 Fc (R&D, 110-HG-100) were PE-conjugated per manufacturer instructions (Abcam) and titrated prior to use. For individual NKG2D-ligand detection the following mAbs were used: MICA (MBL, AMO1), MICB (MBL, BMO1) (both conjugated per manufacturer instructions, Abcam), ULBP-1 (170818), ULBP2/5/6 (165903), ULBP3 (166510), ULBP4 (709116) (all R&D). Cells were washed and stained in phosphate-buffered saline supplemented with 2% fetal calf serum (Gibco) at 4°C after blocking with FcR blocking reagent (Miltenyi). LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen, L34966) was used for live/dead staining. When applicable, cells were fixed using Cytofix kits (BD). Flow cytometry was performed using 4-Laser M Fortessa Analyzers (BD). Flow cytometric analysis was performed using FlowJo V10 (Tree Star).
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