Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats from the New York Blood Center by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Primary NK cells were extracted from PBMCs by negative immunomagnetic isolation using a NK cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, cells were incubated with NK Cell Biotin-Antibody Cocktail and NK Cell MicroBead Cocktail to label non-NK cells, followed by loading onto a LS Separation column (Miltenyi Biotec). The cell suspension was then placed in the magnetic field of a MACS Separator to separate labeled from non-labeled cells. The isolated human NK cells were subsequently cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 100 U/mL IL-2 at 37°C in 5% CO 2 . The purity of the NK cell population was validated by flow cytometry using PE-Vio 770 anti-human CD56 (1:50; REA196 clone; Miltenyi Biotec) and FITC anti-human CD3 (1:50; REA613 clone; Miltenyi Biotec) antibodies.
Fitc anti human cd3
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
The FITC anti-human CD3 is a fluorescently labeled antibody that binds to the CD3 receptor on the surface of human T cells. It is a tool for the identification and enumeration of T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Nk cell biotin antibody cocktail, by Miltenyi Biotec (2 mentions)
Pe vio 770 anti human cd56, by Miltenyi Biotec (2 mentions)
Rea196 clone, by Miltenyi Biotec (2 mentions)
Rea613 clone, by Miltenyi Biotec (2 mentions)
Nk cell microbead cocktail, by Miltenyi Biotec (2 mentions)
Ls separation column, by Miltenyi Biotec (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Nk cell isolation kit, by Miltenyi Biotec (2 mentions)
Propidium iodide solution, by Thermo Fisher Scientific (1 mentions)
Anti human cd4 vioblue, by Miltenyi Biotec (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Miltenyi Biotec (1 mentions)
Anti human cd8 apc, by Miltenyi Biotec (1 mentions)
7 aad, by BD (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Cd161 bv421, by BioLegend (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
4 protocols using fitc anti human cd3
1
Isolation and Culture of Human NK Cells
2
Isolation and Culture of Primary Human NK Cells
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Human peripheral blood mononuclear cells (PBMCs) were isolated from human buffy coats from the New York Blood Center by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). Primary NK cells were extracted from PBMCs by negative immunomagnetic isolation using a NK cell isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. Briefly, cells were incubated with NK Cell Biotin-Antibody Cocktail and NK Cell MicroBead Cocktail to label non-NK cells, followed by loading onto a LS Separation column (Miltenyi Biotec). The cell suspension was then placed in the magnetic field of a MACS Separator to separate labeled from non-labeled cells. The isolated human NK cells were subsequently cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin and 100 U/mL IL-2 at 37°C in 5% CO2. The purity of the NK cell population was validated by flow cytometry using PE-Vio 770 anti-human CD56 (1:50; REA196 clone; Miltenyi Biotec) and FITC anti-human CD3 (1:50; REA613 clone; Miltenyi Biotec) antibodies.
3
Flow Cytometric Analysis of T Cell Subsets
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Analysis of surface molecules on CD3+ T cells was performed using the following monoclonal antibodies: APC-H7-conjugated anti-human CD3 (BD Biosciences, Heidelberg, Germany), anti-human CD4-VioBlue, anti-human CD3-FITC and anti-human CD8-APC (all Miltenyi Biotec GmbH). Immunofluorescence staining of cell surface molecules was performed using the relevant antibodies according to the provider’s instructions. After the staining procedure, cells were washed and evaluated by BD LSRFortessa™ flow cytometer or MACSQuant Analyzer 10 (Miltenyi Biotec GmbH). Before measurement, 7-AAD (BD Biosciences), DAPI (Miltenyi Biotec GmbH) or propidium iodide solution (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added for live/dead discrimination.
4
Immune Cell Phenotyping by Flow Cytometry
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Cells were stained with the following antibodies: anti-human ROR-γt-BV421 (BD Biosciences) (1:20), anti-human CCR6-Alexa 647 (BD) (1:20), anti-human IL-1R1-PE (RnD) (1:20), anti-human CD4-PECy7 (Beckman Coulter) (1:100), CD161-BV421 (Biolegend, San Diego, CA, USA) (1:40), anti-human CD3 FITC (Miltenyi) (1:100). Samples were analysed using Cytoflex cytometer (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo-10 software version 10.3.0.
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