Purified NK cells (2.5×105) were cultured alone or with breast cancer cell lines in 96-well plates in a total volume of 200 μl. Monensin (1μM, eBioScience) was added to cultures. Cells were stimulated with tumor and Trastuzumab with the presence of different mAbs for 16 h at 37 °C with 5% CO2. Cells stimulated with phorbol-12-myristate-13-acetate (PMA) (100 ng/ml; Sigma) and ionomycin (1μM, Sigma) were used as positive controls. After incubation, cells were collected and blocked with human serum 100μl for 15 min at 4 °C. Then cells were labeled with PE-conjugated anti-CD56 (BD Pharmingen) antibodies for 30 min at 4 °C. After cell surface staining, cells were fixed and permeabilized with Fixation and Permeabilization Buffer (BD Pharmingen) and stained with APC-conjugated anti-IFN-γ antibody (BD Pharmingen). After washing with the staining buffer, the cells were resuspended in 300 μl cold staining buffer, followed by analysis with flow cytometry.
Pe conjugated anti cd56
Manufactured by BD
Sourced in United States
PE-conjugated anti-CD56 is a fluorescently labeled antibody that binds to the CD56 cell surface antigen. CD56 is expressed on natural killer cells and a subset of T cells. The PE (phycoerythrin) fluorescent dye allows for the detection and analysis of CD56-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Pe conjugated anti cd69, by BD (3 mentions)
Percp conjugated anti cd3, by BD (3 mentions)
Fitc conjugated anti cd3, by BD (2 mentions)
Navios flow cytometer, by Beckman Coulter (2 mentions)
Percp conjugated anti cd45, by BD (2 mentions)
Apc cy7 conjugated anti cd3, by BD (2 mentions)
Pe cy7 conjugated anti tnf α, by BD (2 mentions)
Fitc conjugated mouse igg isotype, by BD (2 mentions)
Fixation and permeabilization buffer, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti ifn γ antibody, by BD (1 mentions)
Facscanto 2 flow cytometer, by FlowJo (1 mentions)
Human negative selection nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Facscan, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Percp conjugated anti cd19, by BD (1 mentions)
7 protocols using pe conjugated anti cd56
1
Evaluating NK Cell Cytotoxicity Against Breast Cancer
2
Cardiac Differentiation of Embryoid Bodies
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EBs were dissociated on D4 using trypsin-EDTA (0.05%) and incubated with the surface marker PE-conjugated anti-CD56 (1:25 in 0.5% PBS/BSA, BD cat. 347,747) and 1 μg/μL DAPI. On D9, cells were disaggregated with trypsin-EDTA (0.05%) for 5 min and resuspended in PBS to evaluate eGFP expression. On D15, EBs were disaggregated using 1 mg/mL collagenase I for 16 h and trypsin-EDTA (0.05%) for 5 min, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and incubated with anti-troponin T antibody (1:100 in 0.5% PBS/BSA, cardiac isoform Ab-1, Thermo Scientific™, cat. #MS-295-P0) followed by Pacific Blue-conjugated anti-mouse antibody (11000). Analyses were carried out using a FACSCanto II flow cytometer and FlowJo software.
3
Isolation and Characterization of Resting NK Cells
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Untouched resting CD3−CD56+ NK cells were isolated from the thawed CD14− peripheral blood lymphocyte fraction, which was frozen at day -7 in freezing medium consisting of Roswell Park Memorial Institute 1640 (RPMI; Invitrogen, Ref: 21875-034) supplemented with 20% FBS and 10% dimethyl sulfoxide (Sigma-Aldrich, Ref: D2650), after overnight resting at 37°C. NK cells were isolated using the human negative selection NK-cell isolation kit (Miltenyi Biotec, Ref: 130-092-657) according to the manufacturer's instructions. NK-cell purity and viability was at least 95% as determined on a FACScan flow cytometer (BD) following staining with fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (BD; Ref: 21810033 and phycoerythrin (PE)-conjugated anti-CD56 (BD; Ref: 345812) monoclonal antibodies (mABs), and propidium iodide (PI; Invitrogen, Ref: P3566), respectively.
4
Multicolor Flow Cytometry Immunophenotyping
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Blood samples were immunostained with the following panel of antibodies: APC-conjugated anti CD3, FITC-conjugated anti CD4, PE-conjugated anti CD69, PECy5-conjugated anti CD8, PE-conjugated anti CD28, PerCP-conjugated anti CD3, FITC-conjugated anti CD16, PE-conjugated anti CD56, PerCP-conjugated anti CD19, FITC-conjugated anti CD25, PE-conjugated anti Foxp3 and APC-conjugated anti CD4 (all reagents from BD Pharmingen, San Diego, USA). Optimal antibody concentrations were previously defined by titration. For intracellular Foxp3 staining, samples were first stained for CD4 and CD25, then fixed and permeabilized with human Foxp3 buffer set (BD Pharmingen, San Diego, USA) according to manufacturer´s protocols. Briefly, cells were washed twice with permeabilization buffer and then incubated with anti-human Foxp3 at room temperature for 30 min in the dark, before being resuspended in PBS and analyzed.
Flow cytometry data was collected on a FACSCalibur flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis CellQuestPro software (Becton–Dickinson) was used.
Flow cytometry data was collected on a FACSCalibur flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis CellQuestPro software (Becton–Dickinson) was used.
5
B-cell and NK-cell Immunophenotyping in ABO-I KT
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In ABO-I KT and LT recipients, the proportion of peripheral blood B-cell and NK-cell subsets was determined at predesensitization; immediately before transplantation; 1 and 2 weeks after rituximab administration; and 1, 3, 6, 9, and 12 months after transplantation.
For B-cell phenotyping, peripheral blood mononuclear cells (PBMCs) were stained with fluorescein isothiocyanate (FITC)conjugated anti-IgM (BD Pharmingen, San Diego, CA, USA) and PE-conjugated anti-CD19 (BD Pharmingen) monoclonal antibodies (mAbs). B-cells were defined as lymphocytes with both IgM-and CD19-positive phenotypes. For phenotyping NK cells, PBMCs were stained with FITC-conjugated anti-CD3 (BD Pharmingen) and PEconjugated anti-CD56 (BD Pharmingen) mAbs. NK cells were defined as lymphocytes with CD3-negative and CD56-positive phenotypes. Dead cells were excluded from the analysis by light scattering and/or propidium iodide staining. Flow cytometric (FCM) analyses were performed on a FACSCalibur Ò dual-laser cytometer (BD Biosciences, Mountain View, CA, USA). Representative dot plots for B-cells and NK cells are shown in Supplemental Fig. 1.
For B-cell phenotyping, peripheral blood mononuclear cells (PBMCs) were stained with fluorescein isothiocyanate (FITC)conjugated anti-IgM (BD Pharmingen, San Diego, CA, USA) and PE-conjugated anti-CD19 (BD Pharmingen) monoclonal antibodies (mAbs). B-cells were defined as lymphocytes with both IgM-and CD19-positive phenotypes. For phenotyping NK cells, PBMCs were stained with FITC-conjugated anti-CD3 (BD Pharmingen) and PEconjugated anti-CD56 (BD Pharmingen) mAbs. NK cells were defined as lymphocytes with CD3-negative and CD56-positive phenotypes. Dead cells were excluded from the analysis by light scattering and/or propidium iodide staining. Flow cytometric (FCM) analyses were performed on a FACSCalibur Ò dual-laser cytometer (BD Biosciences, Mountain View, CA, USA). Representative dot plots for B-cells and NK cells are shown in Supplemental Fig. 1.
6
Multiparameter Flow Cytometry Analysis
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The following mAbs and reagents were used in this study: Allophycocyanin (APC)-conjugated anti-CD3, APC-conjugated anti-IL-4, APC-conjugated anti-TCRVα24-Jα18, APC-Cy7-conjugated anti-CD3, FITC-conjugated anti-CD3, FITC-conjugated anti-CD45, FITC-conjugated anti-CD56, FITC-conjugated anti-TCRγδ, FITC-conjugated anti-IFN-γ, PE-conjugated anti-TCRVα24-Jα18, PE-conjugated anti-CD45, PE-conjugated anti-CD56, PE-conjugated anti-CD69, PE-conjugated anti-IL-17A, PE-conjugated anti-lymphocyte-activation gene 3 (anti-LAG3), PE-Cy5-conjugated anti-CD161, PE-Cy7-conjugated anti-TNF-α, PerCP-conjugated anti-CD3, PerCP-conjugated anti-CD45, FITC-conjugated mouse IgG isotype, PE-conjugated mouse IgG isotype and PE-Cy7-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA, USA); PE-conjugated anti-programmed death-1 (anti-PD-1; eBioscience, San Diego, CA, USA) and APC-conjugated anti-TCR Vα7.2 (BioLegend, San Diego, CA, USA). Cells were stained with combinations of appropriate mAbs for 20 min at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.5a; Beckman Coulter, Brea, CA, USA).
7
Multiparameter Flow Cytometry Immunophenotyping
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This study used the following mAbs and reagents: Allophycocyanin (APC)-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-conjugated anti-CD69, and fluorescein isothiocyanate (FITC)-conjugated anti-CD45, FITC-conjugated anti-CD56, FITC-conjugated anti-CD107a, FITC-conjugated anti-IFN-γ, phycoerythrin (PE)-conjugated anti-CD3, PE-conjugated anti-CD56, PE-conjugated anti-CD69, PE-Cy7-conjugated anti-TNF-α, PerCP-conjugated anti-CD3, PerCP-conjugated anti-CD45, FITC-conjugated mouse IgG isotype and PE-conjugated mouse IgG isotype control (all from Becton Dickinson, San Diego, CA). Cells were stained with combinations of appropriate mAb for 20 minutes at 4°C. Stained cells were analyzed on a Navios flow cytometer using Kaluza software (version 1.1; Beckman Coulter, Brea, CA).
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