Cryopreserved PBMC (2 × 106) were stimulated for 6 h in 1ml of complete RPMI (RPMI-1640 supplemented with 10% FBS, 100mg/ml streptomycin and 100U/ml penicillin) in the presence of Golgiplug (1µl/ml; BD) and Golgistop (1µl/ml; BD), CD107a (4µl/ml; Biolegend), and CEF (CMV-EBV-Flu) peptide mix (3µg/ml) or Staphyloccal enterotoxin B (SEB; 1µg/ml) as a positive control. When indicated, cells were also stimulated with anti-SLAMF7 mAb or a mouse IgG2b Isotype control antibody (5µg/ml, Biolegend), and a goat anti-mouse IgG cross-linker (EMD Millipore). At the end of the stimulation, cells were stained for live-dead cells (Zombie NIR Fixable Viability Kit, Biolegend), then stained for surface antibodies with CD3 (BD Horizon BUV395 anti-human CD3; BD), CD4 (PerCP eFluor 710 anti-human CD4; eBioscience), CD8 (PerCP anti-human CD8; Biolegend), fixed / permeabilized (Cytofix/cytoperm; BD) and stained for IFNγ (Alexa Fluor 647 anti-human IFNγ; Biolegend). Cells were then acquired on an LSRII SORP and analyzed by using FlowJo (version 10.1r5, FlowJo Enterprise).
Alexa fluor 647 anti human ifnγ
Manufactured by BioLegend
Sourced in United States
Alexa Fluor 647 anti-human IFNγ is a fluorescently labeled antibody that binds to human interferon gamma (IFNγ). It can be used to detect and quantify IFNγ in various immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Percp anti human cd8, by BioLegend (1 mentions)
Zombie nir fixable viability kit, by BioLegend (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Novocyte quanteon, by Agilent Technologies (1 mentions)
Pe antihuman tnf α, by BioLegend (1 mentions)
Bv605 anti mouse cd4, by BioLegend (1 mentions)
2 protocols using alexa fluor 647 anti human ifnγ
1
Cryopreserved PBMC Stimulation Assay
2
Murine T-cell Phenotyping by Flow Cytometry
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For flow cytometry analysis, single-cell suspensions were stained with the following antibodies: BV605 anti-mouse CD4 and Alexa Fluor 700 CD8 (Biolegend, San Diego, CA, USA). For intracellular staining, cells were stimulated for 4 h at room temperature with PMA (50 ng/mL), ionomycin (1 µg/mL), and Brefeldin A. Following fixation and permeabilization, cells were stained with Alexa Fluor 647 anti-human IFN-γ and PE anti-human TNF-α (Biolegend, San Diego, CA, USA). Cells were acquired with a NovoCyte Quanteon (Agilent Technologies, Santa Clara, CA, USA), and data analysis was performed with FlowJo software (Version 10.8, FlowJo, Ashland, OR, USA).
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