NK cell phenotype of melanoma patients enrolled in the trial was examined using fluorochrome-conjugated antibodies against the following cell-surface markers: CD56-FITC, CD3-PC7, CD16-APC, CD69-BV421, NKp30-BV711, CXCR3-BV421, CCR3-BV510 (BD Biosciences; San Diego, CA), NKp44-PerCP eFluor 710 (eBioscience; San Diego, CA), CXCR1-PE (R&D Systems; Minneapolis, MN), CCR7-BV711 (BioLegend; San Diego, CA), and matching IgG isotype controls from the same vendors. The immune checkpoint and NK cell activation receptor panel included the following markers: Zombie NIR Fixable Viability Dye (BioLegend; San Diego, CA), CD3-PE-Vio770 (Miltenyi Biotec; San Diego, CA), ANK-1-PE (Santa Cruz Biotechnology; Dallas, TX), TIGIT-PerCP eFluor 710 (eBioscience), CD45-BUV395, CD56-BV510, CD16-BUV737, NKG2D-APC, NKp46-BV711, CD69-BV421, and PD-1-BV650 (BD Biosciences).
Cd3 pe vio770
Manufactured by Miltenyi Biotec
The CD3-PE-Vio770 is a fluorochrome-conjugated monoclonal antibody that binds to the CD3 antigen, a key component of the T-cell receptor complex. It is commonly used in flow cytometry applications to identify and quantify T cells in biological samples.
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Lab products found in correlation
Cd16 apc, by BD (2 mentions)
Cd56 fitc, by BD (2 mentions)
Nkp30 bv711, by BD (2 mentions)
Cxcr3 bv421, by BD (2 mentions)
Nkp44 percp efluor 710, by Thermo Fisher Scientific (2 mentions)
Cxcr1 pe, by R&D Systems (2 mentions)
Cd56 bv510, by BD (2 mentions)
Cd16 buv737, by BD (2 mentions)
Cd69 bv421, by BD (2 mentions)
Cd8 fitc, by Miltenyi Biotec (2 mentions)
Nkg2d apc, by BD (1 mentions)
Tigit percp efluor710, by Thermo Fisher Scientific (1 mentions)
Cd3 pc7, by BD (1 mentions)
Ccr7 bv711, by BioLegend (1 mentions)
Zombie nir fixable viability dye, by BioLegend (1 mentions)
Pd 1 bv650, by BD (1 mentions)
Cd3 pe cy7, by BD (1 mentions)
Cd45 buv395, by BD (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Ccr7 bv510, by BD (1 mentions)
6 protocols using cd3 pe vio770
1
Comprehensive NK Cell Profiling in Melanoma
2
Multiparametric Characterization of NK Cells
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One-step staining of cell-surface antigens was performed using fluorochrome-conjugated primary antibodies as previously described (10 (link), 18 (link), 33 (link)). For the analysis of blood NK cells two antibody panels were constructed around CD56-FITC, CD16-APC, and CD3-PE-Cy7 (BD Bioscience, San Jose, CA) antibodies. NK cell activation receptors were evaluated with CD69-BV421, NKp30-BV711 (BD Bioscience), and NKp44-PerCP eFluor 710 (eBioscience, San Diego, CA) antibodies. Chemokine expression levels were tested using CXCR1-PE (R&D Systems, Minneapolis, MN), CXCR3-BV421, and CCR7-BV510 (BD Bioscience) antibodies. For the TINK analysis, Zombie NIR (BioLegend), CD45 BUV395, CD56 BV510, CD16 BUV737 (BD Bioscience), and CD3 PE-Vio770 (Miltenyi Biotec) antibodies were used. Suitable IgG controls were acquired from the same vendors. FACS analyses were performed using the BD LSRFortessa™ cell analyzer, and analyzed using FlowJo v10 (FlowJo, LLC; Ashland, OR) software.
3
Isolation of T Cell Subsets
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Following respirometry, cell suspensions were centrifuged and PBMC were resuspended in cell separation buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA; Miltenyi Biotec, Bergisch-Gladbach, Germany) for the separation of T cell subsets. Appropriate volumes of antibodies (CD4-APC [10 µl/107 cells], CD8-FITC [10 µl/107 cells], CD45RA-PE [5 µl/107 cells], CD3-PE-Vio770 [5 µl/107 cells], all purchased from Miltenyi Biotec) were added and cells were stained according to the manufacturer’s protocol. Naïve T helper cells (CD3+CD4+CD45RA+), memory T helper cells (CD3+CD4+CD45RA−), naïve cytotoxic T cells (CD3+CD8+CD45RA+), and memory cytotoxic T cells (CD3+CD8+CD45RA−) were then isolated by fluorescent-activated cell sorting on a BD FACSAria III cell sorter (BD Biosciences, Heidelberg, Germany). Propidium iodide staining was used to distinguish dead from living cells.
4
Mouse T Cell Absolute Counts from Mandibular Vein
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T cell absolute count was performed on blood samples kept from the mandibular vein of the mouse.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
For the phenotypic characterization of cell populations, the following antibodies were used: CD8-FITC (Miltenyi Biotec), CD25-APC (Pharmingen), CD3-PE-Vio770 (Miltenyi Biotec), CD4-APC-Vio770 (Milteny Biotec), CD45R (B220)-Violblu (Milteny Biotec), NK1.1-PE (Milteny Biotec).
At predetermined optimal concentrations, 100 μl of blood was stained by incubation with the antibodies. Fifty microliters of CountBright Absolute Counting Beads (Molecular Probes) was added, and, following lysis of red blood cells, cells were acquired on a CyAn Cytometer (Beckman Coulter). By comparing the ratio of bead events to cell events, absolute numbers of cells in the sample were calculated.
Some experiments were performed by acquiring the stained blood samples on the CytoFLEX cytometer (Coulter), equipped with a volumetric sample injection module, which enables volumetric sampling and provides absolute cell counts for all samples without the use of beads.
5
Multiparametric Analysis of T-cell Subsets
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Foxp3-APC (ebioscience, clone 236A/E7) and mIgG1 κ APC isotype control (ebioscience, clone P3.6.2.8.1), CD25-PE (Miltenyi), CTLA-4-BV421 (BD Biosciences, clone BNI3) and mIgG2a κ BV421 isotype control (BD Biosciences), IFN-γ-PE and mIgG1 κ PE isotype control (ebioscience), IFN-γ-FITC and mIgG1 κ FITC isotype control (ebioscience), IL-17A eFluor450 and mIgG1 κ eFlour450 isotype control (ebioscience), CD45RA-PE-Vio770 (Miltenyi), CD45RA-FITC (Miltenyi), CD45RO-PE (BD Biosciences), CD4-PerCP (BD Biosciences), CD3-PE-Vio770 (Miltenyi), CD8-eFlour450 (ebioscience), fixable viability dye-eFlour780 (ebioscience), CFSE (Molecular Probes).
6
Flow Cytometry Immune Cell Profiling
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Anti-VISTA antibodies and isotype control (Human IgG1 Isotype control, Bio X Cell BP0297) were labeled with Alexa Fluor 647 using the protocol in the conjugation kit (Biotium). Monocytes, T-cells, and Neutrophils were labeled in a human blood sample (BIOIVT) by flow cytometry using CD45-VioGreen (130-110-638), CD3-PE-Vio 770 (130-113-140), CD16-Vio Bright B515 (130-119-616) and CD14-PE (130-113-147) in the presence of FcR Blocking Reagent (130-059-901; all reagents from Miltenyi Biotec and antibodies diluted 50-fold as per manufacturers recommendation). Samples with appropriate FMO (Fluorescence Minus One) controls for each antibody were analyzed in parallel. RBC were lysed using Lysis Buffer (BD Biosciences 555899). Samples were washed in 1x PBS pH7.4 containing 1% heat inactivated fetal bovine serum, and propidium iodide (PI) staining (Miltenyi 130-093-233) for Live/dead cell discrimination done immediately before analysis using a MACS Quant Analyzer (Miltenyi). For NK cells, isolated human NK cells (Hemacare/Charles River Labs) were stained with labeled anti-VISTA or isotype control mAbs and CD56-FITC (Miltenyi 130-114-740; 50-fold diluted) in MACSQuant Running Buffer (Miltenyi 130-092-747), and Sytox Blue (Thermo Fisher Scientific S34862) was used for gating for live cells.
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