Single-cell suspensions were stained with the appropriate monoclonal antibody in PBS containing 2% FCS. When necessary, intracellular staining was performed by use of the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Fortessa, FACS Verse, and ARIAIII (BD Biosciences) were used for cell sorting and analysis. Antibodies specific for NK1.1 (PK136; 1:100), CD19 (1D3; 1:400), CD3 (17A2; 1:400 or REA641; Miltenyi Biotec; 1:150); CD122 (TM-β1; 1:200), NKp46 (29A1.4; 1:100), KLRG1 (2F1; 1:200), CD27 (SB/199; 1:200), CD11b (M1/70; 1:200), IL-7R (A7R34; eBioscience; 1:200) CD49b (DX5; 1:100), CD49a (Ha31/8; 1:200) Ly49H (3D10; 1:200) Ly49D (4E5; 1:200), NKG2D (C4; 1:200), NKG2A/C/E (20d5; 1:200), Ly49C/I (5e6; 1:100), CD107a (104B; 1:100), and IFN-γ (XMG1.2; 1:100), DNAM-1 (10E5; 1:200); Ki-67 (AF488; 1:50) were from BD Pharmingen unless stated otherwise.
Dnam 1
Manufactured by BD
Sourced in United States
DNAM-1 is a laboratory equipment designed for the detection and analysis of deoxyribonucleic acid (DNA). Its core function is to provide accurate and reliable measurements of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Cd107a, by BD (2 mentions)
Klrg1, by Thermo Fisher Scientific (2 mentions)
Nkg2d, by BD (2 mentions)
Rea641, by Miltenyi Biotec (1 mentions)
Il 7r a7r34, by Thermo Fisher Scientific (1 mentions)
Nkg2a c e, by BD (1 mentions)
Ki 67 af488, by BD (1 mentions)
Cd49b, by Thermo Fisher Scientific (1 mentions)
Ly49c 1, by BD (1 mentions)
Ifn γ xmg1.2, by BD (1 mentions)
Cd11b, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Aria 3, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Tigit, by Thermo Fisher Scientific (1 mentions)
Nkg2a, by Miltenyi Biotec (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Gallios instrument, by Beckman Coulter (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
6 protocols using dnam 1
1
Comprehensive Multicolor Flow Cytometry Protocol
2
Comprehensive Immune Phenotyping of Blood Samples
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Blood samples (100 µl) collected from the participants were incubated for 25 min at 25 °C in a flow cytometry tube with the following specific antibodies (5 µl each): CD3 (563219, BD), CD16 (302018, Biolegend), CD56 (555518, BD), TIGIT (12-9500-42, eBioscience), DNAM-1 (559788, BD), NKG2A (130-113-565, Miltenyi Biotec), LAIR-1 (ab27744, Abcam), Siglec-7 (ab200558, Abcam), and KLRG1 (25–9488-42, eBioscience). In addition, an isotype control was prepared for each sample. After incubation, the reaction mixture in each tube was mixed thoroughly with 2 ml of hemolysin. The samples were then washed for flow cytometry analysis (BD FACSCanto™ Flow Cytometer) within 4 h.
3
Multiparameter Flow Cytometry of Immune Cells
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Mouse anti-human mAbs against CD73, CD56, CD69, NKp44, NKp30, NKp46, NKG2D, DNAM-1 and granzyme B were purchased from BD Biosciences (San Jose, CA, USA), mouse anti-human mAbs against CD105, CD90, CD13, CD44, CD31, CD34, CD45 and CD3 were purchased from eBioscience (San Diego, CA, USA) and mAbs against TNF-α, IFN-γ and perforin were obtained from Beckman Coulter (Fullerton, CA, USA) and Biolegend (San Diego, CA, USA).
The cells were collected, washed, and resuspended in PBS supplemented with 1% heat-inactivated FBS. Thereafter, the cells were directly stained extracellularly with 2 μg mAb in a total staining volume of 100 μL per 106 cells. The cells were stimulated at 37°C for 4 hours with Leukocyte Activation Cocktail (BD Pharmingen, San Jose, CA, USA) and fixed and permeabilized prior to intracellular staining using Pharmingen's staining protocol. The data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software.
The cells were collected, washed, and resuspended in PBS supplemented with 1% heat-inactivated FBS. Thereafter, the cells were directly stained extracellularly with 2 μg mAb in a total staining volume of 100 μL per 106 cells. The cells were stimulated at 37°C for 4 hours with Leukocyte Activation Cocktail (BD Pharmingen, San Jose, CA, USA) and fixed and permeabilized prior to intracellular staining using Pharmingen's staining protocol. The data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software.
4
Redirected Cytotoxicity Assay for NK Cells
5
Phenotypic Characterization of NK-92 Cells
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For flow cytometry, NK-92 cells were stained with monoclonal antibodies specific for CD56, CD3, CD45, CD28, CD16, CD11a, CD2, NKG2D, NKp30, NKp46, DNAM-1, CD95 (BD Bioscience) for 30 minutes on ice. CAR expression on the surface of NK-92 cells was detected with an anti-mouse F(ab’)2 antibody (Jackson Immunoresearch). For checkpoint molecules, antibodies specific for PD-1, LAG-3 and TIM-3 (BD Biosciences) were used. Dead cells were excluded using 7-AAD viability dye (BD Biosciences). Samples were acquired using an LSR-Fortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo 10 software (BD Biosciences).
6
Comprehensive Immune Phenotyping of PBMC
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Surface marker staining of PBMCs from patients’ fresh blood samples for the following markers was performed in PBS at 4°C for 30 min following Fc blockade: CD3 (BD, UCHT1), CD56 (BD, NKCM16), CD335 (NKp46) (BD, 9E2/NKp46), CD337 (NKp30) (BD, p30-15), CD57 (BD, NK-1), DNAM1 (BD, DX11), NKG2C (CD159c) (Miltenyi Biotec, REA205), NKG2A (CD159a) (Miltenyi Biotec, REA110), NKG2D (BD, 1D11), KIR2DL1 (BD, HP-3E4), KIR2DL2/L3 (Beckman Coulter), KIR3DL1 (Miltenyi Biotec, REA168), CD107a (BD, H4A3), and IFN-γ (BioLegend, B27). Acquisition was performed on an LSR Fortessa instrument (BD Biosciences), and Flowjo software (TreeStar) was used for analysis. Single-stain controls for compensation were generated utilizing UltraComp eBeads (eBioscience, BioLegend, and Miltenyi Biotec), and the analysis of acquired samples was performed in FlowJo (FlowJo, LLC, Ashland, CA) data analysis software. Data were gated using FlowJo (FlowJo, LLC). Following data acquisition, the channel intensity was normalized using calibration beads. Before the experiment, we verified the configuration by selecting an appropriate configuration and defining a new baseline every 24 h. We set up and ran a performance check. The anti-human monoclonal antibodies used for flow cytometry are listed in Supplementary Table 1
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