NK cell degranulation upon co-incubation with target cells was determined by the expression of CD107a on the cell surface, which serves as a surrogate marker for NK cell degranulation [58 (link)]. In brief, overnight cultured NK cells enriched with the EasySep human NK cell enrichment kit (StemCell Technologies) from PBMCs from KIR2DL5A*001-positive donors or donors lacking KIR2DL5 genetically were co-cultured with CD155-expressing (CD155-transduced 721.221) or CD155-nonexpressing (721.221) target cells at an effector to target ratio of 1:2 in 200 μl complete R10 for 4 h at 37°C. During co-incubation, each well contained 2 μl anti-CD107a (clone LAMP-1, Biolegend) and 25μl/ml Brefeldin A. Cells were subsequently stained with LIVE/DEAD Fixable Near-IR and with the following antibodies: anti-CD3-BUV395 (clone UCHT1, BD), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-CD56-BV785 (clone NCAM16.1, BD), anti-KIR2DL5-PE (clone UP-R1, Biolegend) for 15 min at RT and fixed with CellFix (BD) before flow cytometric acquisition.
Anti cd3 buv395 clone ucht1
Manufactured by BD
Anti-CD3-BUV395 (clone UCHT1) is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen on human T cells. It can be used for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellfix, by BD (2 mentions)
Live dead fixable near ir, by Thermo Fisher Scientific (2 mentions)
Easysep human nk cell enrichment kit, by STEMCELL (1 mentions)
Live dead fixable near ir, by BD (1 mentions)
Anti cd16 pe cy7 clone 3g8, by BioLegend (1 mentions)
Anti cd4 bv711 clone rpa t4, by BioLegend (1 mentions)
Anti p24 fitc clone kc57, by Beckman Coulter (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Anti cd3 bv605 clone okt3, by BioLegend (1 mentions)
Anti cd56 pe cy7 clone ncam16, by BD (1 mentions)
Anti mip 1β percp cy5, by BD (1 mentions)
Anti ifn γ v450, by BD (1 mentions)
Anti cd69 bv421 clone fn50, by BioLegend (1 mentions)
4 protocols using anti cd3 buv395 clone ucht1
1
Quantifying NK Cell Degranulation
2
Flow Cytometric Analysis of HIV-1 Infection
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To assess cell surface expression of proteins, flow cytometry was performed. Cells were washed with PBS and subsequently stained with the viability dye LIVE/DEAD Fixable Near-IR (Life Technologies) and with the antibodies anti-CD3-BUV395 (clone UCHT1, BD), anti-CD4-BV711 (clone RPA-T4, Biolegend), anti-CD155-PE (clone SKIL4, Biolegend), anti-HLA-ABC-Pe-Cy7 (clone W6/32, Biolegend), anti-HLA-E-BV421 (clone 3D12, Biolegend), anti-tetherin-APC (clone RS38E, Biolegend) and anti-IgG1-PE isotype control (clone MOPC21, Biolegend) for 15 min at RT. After washing the cells with PBS, an intracellular staining was performed. In brief, cells were incubated in BD Cytofix/Cytoperm for 20 min at 4°C, washed with BD Perm/Wash buffer and stained with anti-p24-FITC (clone KC57, Beckman Coulter) for 20 min. After another washing step, cells were fixed in BD Cellfix and analyzed by flow cytometry (BD LSR Fortessa). HIV-1-infected cells were defined as p24 Gag+ CD4dim and uninfected as p24 Gag- CD4+ cells. Cells infected with HIV-1 ΔNef mutant viruses were defined as p24 Gag+ and tetherin- cell, as Nef-deficient viruses are not able to downregulate CD4.
3
Comprehensive Immune Profiling using Multicolor Flow Cytometry
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The following anti-human antibodies were used in this study: anti-CD3-BV605 (clone OKT3, Biolegend), anti-CD3-BUV395 (clone UCHT1, BD Bioscience), anti-CD14-V500 (clone M5E2, BD Bioscience), anti-CD19-BV510 (clone SJ25C1, BD Bioscience), anti-CD56-PE-Cy7 (clone NCAM16.2, BD Bioscience), anti-CXCR6-PE or anti-CXCR6-APC (clone K041E5, Biolegend), anti-CD57-AF488 (clone TB01, eBioscience), anti-TRAIL-BV421 (clone RIK-2, BD Bioscience), anti-CD49a-FITC (clone TS2/7, Biolegend), anti-HLA-DR V500 (clone G46-6, BD Bioscience) and anti-CD69-PE-Dazzle (clone FN50, Biolegend) for surface antigens; and anti-T-bet-eFlour610 (clone 4B10, eBioscience) and anti-Eomes-PE-eFlour610 (clone Dan11mag, eBioscience) for intranuclear antigens; anti-IFNγ-V450 (BD Bioscience), anti-MIP-1β-PerCP-Cy5.5 (cloneD21–1351, BD Bioscience), anti-GM-CSF-PE-CF594 (clone BVD2–21C11, BD Bioscience), anti-TNF-FITC (clone MAb11, BD Bioscience), anti-Granzyme B-AlexaFlour700 (clone GB11, BD Bioscience) and anti-Perforin-PerCP-Cy5.5 (clone dG9, Biolegend) for intracellular antigens.
4
Evaluating KIR-Mediated Activation
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Streptavidin Dynabeads were coated with biotinylated PVR (CD155), Nectin-2 (CD112), biotin, anti-KIR2DL1, anti-KIR2DL3, anti-KIR2DL5 and anti-KIR3DL1 as described before. 2.5 x 104 cells of each reporter cell line were seeded into a well of a tissue culture-treated 96-well plate and co-incubated with 10 μl of the protein-coated beads for 5 h at 37°C/5% CO2 in a final volume of 200 μl. For blocking experiments, prior to co-incubation with beads, KIR2DL5+ JRC were blocked for 30 min with 30 μg/ml purified anti-KIR2DL5 or 30 μg/ml purified IgG1 isotype control antibody. Blocking antibodies remained in the wells during the whole assay. After co-incubation, cells were washed with PBS and stained with the viability dye LIVE/DEAD Fixable Near-IR (Life Technologies), anti-CD3-BUV395 (clone UCHT1, BD Biosciences), anti-CD69-BV421 (clone FN50, Biolegend) and the appropriate KIR antibody conjugated to PE (anti-KIR2DL1-PE (clone REA284), anti-KIR2DL3-PE (clone REA147), anti-KIR2DL5-PE (clone UP-R1), anti-KIR3DL1-PE (clone DX9) (Miltenyi). Cells were fixed in CellFix (BD Biosciences) and CD69 expression as a readout for KIR crosslinking was analyzed by flow cytometry.
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