Peripheral blood lymphocytes (PBLs) from HSCT recipients were cocultured with pamidronate-pretreated autologous or allogeneic (refers to third-party healthy subjects) DCs at the ratio of 2:1, at a final of 3 × 105 cells/well in 96-well plates. In some experiments, the neutralization antibody anti-NKG2D (10 μg/ml, BioLegend, USA) was used for blocking NKG2D during coculture. After 7 and 14 days of coculture, the percentages and differentiation and activation phenotypes of Vδ2+ T cells were detected by flow cytometry. Different combinations of monoclonal antibodies allowed identifying the differentiation profile of Vδ2+ T cells by the expression of CD45RO and CD27 (Naive: CD45RO−CD27+, Central Memory (CM): CD45RO+CD27+, Effector Memory (EM): CD45RO+CD27− and terminal differentiation (TD): CD45RO−CD27−). For cell differentiation and activation assay, the cultured cells were stained with fluorochrome-labeled anti-CD27, anti-CD45RO, anti-HLA-DR, anti-CD38, and anti-NKG2D antibodies (BioLegend, USA).
Anti nkg2d
Manufactured by BioLegend
Sourced in United States
Anti-NKG2D is a laboratory reagent that binds to the NKG2D receptor expressed on natural killer (NK) cells and some T cells. NKG2D is an activating receptor that recognizes stress-induced ligands and plays a role in immune cell function. Anti-NKG2D can be used in research applications to study the role of the NKG2D receptor in cellular processes.
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Lab products found in correlation
Gallios flow cytometer, by Beckman Coulter (5 mentions)
Anti nkp44, by BioLegend (4 mentions)
Anti nkp46, by BioLegend (4 mentions)
Anti cd27, by BioLegend (3 mentions)
Anti nkp30, by BioLegend (3 mentions)
Anti cd69, by BioLegend (2 mentions)
Anti dnam 1, by BioLegend (2 mentions)
Anti cd107a, by BioLegend (2 mentions)
Anti ifn γ, by BioLegend (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Anti cd226, by BioLegend (2 mentions)
Anti cd107a antibody, by BioLegend (2 mentions)
Igg1 isotype control, by BioLegend (2 mentions)
Anti cd2, by BD (2 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Apc conjugated anti mouse cd45 antibody, by BioLegend (1 mentions)
Flow count, by Beckman Coulter (1 mentions)
Facs canto machine, by BD (1 mentions)
Apc cy7 conjugated anti cd45, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
14 protocols using anti nkg2d
1
Vδ2+ T Cell Differentiation Upon DC Coculture
2
Cytotoxicity Assay of NK Cells
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cNK and ML NK cells were incubated with tumor targets at 5:1 ET ratio as previously described.13 (link),14 (link) When indicated, cNK and ML NK cells were pre-incubated with anti-NKG2D, anti-NKp46, anti-NKp30, and anti-DNAM1 (5 µg/mL; all Biolegend) or a combination of anti-NKG2D, anti-NKp46 and anti-DNAM for 30 minutes before co-incubation with tumor targets. Cells incubated with Isotype control were used as control. At the end, the cells were stained with anti-CD56 (clone N901), anti-CD3 (clone UCHT1), anti-NKG2A (clone Z199), and anti-CD45 (clone J33) followed by intracellular staining with anti-IFN-γ (clone B27). Data was acquired in a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star v10.8).
3
Multicolor Flow Cytometric Analysis of NK Cells
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Single mononuclear cell suspensions were stained with antibodies for 30 min at 4 °C in the dark. Cells were then washed with fluorescence-activated cell sorting (FACS) buffer (PBS, 0.5% FBS, 0.05% NaN3) and resuspended in FACS buffer containing propidium iodide (PI) before being subjected to multicolor flow cytometric analysis by a FACS Canto machine (BD Biosciences). The data were analyzed using FACS Diva software (ver. 6.1.3; BD Biosciences).
The antibodies used were as follows: Alexa Fluor® 488-conjugated anti-human natural killer group 2 membrane C (NKG2C) from R&D Systems (Minneapolis, MN, USA) and anti-human CD159a (NKG2A)-PE and anti-human NKp80-PE (Beckman Coulter, Miami, FL, USA). The following antibodies against human antigens were from BioLegend: fluorescein isothiocyanate (FITC)-conjugated anti-CD8a, anti-CD16, anti-CD158a/h, and anti-CD158e1; phycoerythrin (PE)-conjugated anti-CD56, anti-NKp30, anti-NKp44, anti-NKp46, anti-CD57, anti-NKG2D, anti-CD158b, and anti-CD158f; PE-Cy7-conjugated anti-CD3 and anti-CD56; APC-conjugated anti-CD16 and anti-CD94 monoclonal antibodies; and APC-Cy7-conjugated anti-CD45. The APC conjugated anti-mouse CD45 antibody was from BioLegend.
The absolute number of cells was calculated using fluorescent beads (Flow-Count; Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions.
The antibodies used were as follows: Alexa Fluor® 488-conjugated anti-human natural killer group 2 membrane C (NKG2C) from R&D Systems (Minneapolis, MN, USA) and anti-human CD159a (NKG2A)-PE and anti-human NKp80-PE (Beckman Coulter, Miami, FL, USA). The following antibodies against human antigens were from BioLegend: fluorescein isothiocyanate (FITC)-conjugated anti-CD8a, anti-CD16, anti-CD158a/h, and anti-CD158e1; phycoerythrin (PE)-conjugated anti-CD56, anti-NKp30, anti-NKp44, anti-NKp46, anti-CD57, anti-NKG2D, anti-CD158b, and anti-CD158f; PE-Cy7-conjugated anti-CD3 and anti-CD56; APC-conjugated anti-CD16 and anti-CD94 monoclonal antibodies; and APC-Cy7-conjugated anti-CD45. The APC conjugated anti-mouse CD45 antibody was from BioLegend.
The absolute number of cells was calculated using fluorescent beads (Flow-Count; Beckman Coulter, Brea, CA, USA) according to the manufacturer’s instructions.
4
Comprehensive NK Cell Phenotyping
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The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
5
NK Cell Receptor Expression Analysis
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The following FITC‐, PE‐, PE‐Cy5–, or APC‐conjugated mAbs were obtained from BD Bioscience, eBioscience, or Biolegend: anti‐NKG2D, anti‐LFA‐1 (CD11a), anti–DNAM‐1 (CD226), anti‐MICA/B, anti‐ULBP1, anti‐ULBP2, anti‐ULBP4, anti‐ICAM‐1 (CD54), anti‐ICAM‐2 (CD102), anti‐PVR (CD155), and anti‐Nectin‐2 (CD112). Additionally, the following blocking mAbs were also used: anti‐NKG2D (1D11 from eBioscience), anti‐CD11a (HI111 from Biolegend), anti‐CD226 (DX11 from Abcam), and mouseIgG1κ isotype control (P3.6.2.8.1 from eBioscienc). Specific extracellular signal–regulatory kinase1/2 (ERK1/2) inhibitor (PD098059) was purchased from Sigma‐Aldrich.
6
Quantification of NKG2D Ligand Expression on Tumor Cells
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Tumor cell lines were collected and stained with anti-MICA/B-APC (BioLegend), anti-ULBP1-APC, anti-ULBP2/5/6-APC, anti-ULBP3-PE, and anti-ULBP4-APC (R&D Systems, Minneapolis, MN, USA) antibodies in fluorescence-activated cell sorting (FACS) buffer at 4°C for 30 min. The cells were washed twice with FACS buffer, followed by cytometric analysis. Anti-CD3, anti-CD8, anti-CD4, and anti-NKG2D antibodies were employed to test the NKG2D expression levels of T cells; these antibodies were purchased from BioLegend. Anti-CD56 and anti-CD16 antibodies were used to determine the purity of isolated NK cells. Flow-cytometry experiments were performed with an Agilent flow cytometer, a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA, USA), or a C6 flow cytometer (BD Bioscience, East Rutherford, NJ, USA). FlowJo v.10 software was used for data analysis.
7
NK Cell Cytotoxicity Assay
8
Multiparametric Immunophenotyping of Liver-Infiltrating Lymphocytes
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Anti‐mouse antibodies used here include CD1d tetramer (provided by the NIH Tetramer Facility), anti‐CD3, anti‐NK1.1, anti‐Dx5, anti‐NKG2D, anti‐NKG2A, anti‐CD69, anti‐IL‐4, anti‐IFN‐γ, anti‐TNF‐α and anti‐CD107a (from BioLegend) and anti‐Tim‐3 from eBioscience. Briefly, cell membrane markers were directly labelled for 30 minutes in dark at 4°C. For intracellular staining, freshly isolated IHLs were stimulated with or without α‐Galcer for 6 or 2 hours, and Brefeldin A (BFA) was added into the cell culture 4 or 2 hours before harvest. For detecting CD107a expression, fluorescence‐labelled anti‐CD107a was added into cell culture and maintained for the whole incubation process. All samples were tested by BD FACSAria III flow cytometer (BD Biosciences), and FCM data were analysed using BD FACSDiva 7.0 or FlowJo 7.0 software.
9
Functional Characterization of NK Cells
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Control and ML NK cells were restimulated with K562, DM6, M14 cells, or autologous melanoma targets for 6 hours in presence of 1 ng/mL rhIL15. Effector/target (E:T) ratio was 5:1, unless otherwise indicated. When indicated, control and ML NK cells were preincubated with anti-NKG2D (2.5 μg/mL; BioLegend) and anti-NKp46 (2.5 μg/mL; BioLegend) blocking antibodies 30 minutes before coincubation with tumor targets. Cells were stained as described previously (21 ). Data were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software (Tree Star v10.6).
10
NK Cell-Mediated Cytotoxicity Assay
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