Cells were harvested after stimulation and viability of cells post activation was measured using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Surface staining of antigen was performed at 4°C for 20 minutes. For intracellular antigens, cells were fixed and permeabilized using the FOXP3/Transcription factor staining kit (eBioscience), and incubated with the appropriate antibody for 1 hour at 4°C. Acquisition of cells was performed using the BD FACSCantoTM II Flow Cytometer and subsequent analysis was performed using FlowJo (v9 or v10). The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2μg/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFNγ expression was analyzed using the IFN-γ Secretion Assay (PE, Miltenyi Biotec) following manufacturer’s instructions.
Anti sheep igg nl557
Manufactured by R&D Systems
Anti-sheep IgG-NL557 is a secondary antibody conjugated to NL557, a fluorescent dye. It is used to detect and visualize sheep immunoglobulin G (IgG) in various immunological applications such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (2 mentions)
Foxp3 transcription factor staining kit, by Thermo Fisher Scientific (2 mentions)
Defa1, by R&D Systems (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd4 pacific blue, by BioLegend (2 mentions)
Cd45ro fitc, by BioLegend (2 mentions)
Cd45ra apc, by BioLegend (2 mentions)
Cd69 apc, by BioLegend (1 mentions)
Uchl1, by R&D Systems (1 mentions)
2 protocols using anti sheep igg nl557
1
Flow Cytometric Analysis of Activated Cells
2
Viability and Surface Antigen Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were harvested after stimulation and viability of cells post activation was measured using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Surface staining of antigen was performed at 4 C for 20 minutes. For intracellular antigens, cells were fixed and permeabilized using the FOXP3/Transcription factor staining kit (eBioscience), and incubated with the appropriate antibody for 1 hour at 4 C. Acquisition of cells was performed using the BD FACSCantoTM II Flow Cytometer and subsequent analysis was performed using FlowJo (v9 or v10).
The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2 g/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFN expression was analyzed using the IFN-Secretion Assay (PE, Miltenyi Biotec) following manufacturers instruction.
The clone and dilution of antibodies used for flow cytometry were as follows: CD4-Pacific Blue (Biolegend, OKT4, 1:200), CD69-APC (Biolegend, FN50, 1:200), CD45RA-APC (Biolegend, HI100, 1:200), CD45RO-FITC (Biolegend, UCHL1, 1:200), DEFA1 (R&D Systems, 2 g/ml), anti-sheep IgG-NL557 (R&D Systems, 1:500). IFN expression was analyzed using the IFN-Secretion Assay (PE, Miltenyi Biotec) following manufacturers instruction.
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