eBioscience™ from Invitrogen Antibodies
IFN gamma Monoclonal Antibody (4S.B3), Alexa Fluor 488
53-7319-42
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CD4+ and CD8+ T cells produce distinct cytokines tailored to the specific pathogen eliciting the immune response. Production of IFN-gamma is critical for defense against intracellular pathogens. We are interested in the expression of IFN-gamma by various memory T cell subsets found in human peripheral blood. To assay for IFN-gamma production, we stimulate cells with PMA/ionomycin for 3 hours prior to staining, followed by flow cytometric analysis with a multi-color panel to differentiate CD4/CD8+ T cells as well as naive/memory subsets.
Flow Cytometry
human PBMCs
1:100, 30min, 4C
1X eBioscience Perm/Wash Buffer
N/A
BD Fortessa
We obtained PBMCs from freshly donated blood from healthy volunteers. Bulk mononuclear cells were then stimulated with PMA/ionomycin in the presence of brefeldin (Golgi Plug) to induce cytokine production and accumulation. Samples were then fixed and permeablized with the Foxp3 transcription factor staining kit and analyzed by flow cytometry. Within the lymphocyte gate, we gated CD3+ CD8+ T cells for analysis of IFN-gamma. A clearly separable subset of cells had robust IFN-gamma production (Figure shows live bulk CD3+ CD8+ cells). Positive IFN-gamma signal was largely detected in the memory (CD45RA- compartment).
Excellent separation of positive cells; can be multiplexed into large panels; reasonable dilution factor
Requires brefeldin treatment
Strong detection of IFN-gamma production by human T cell subsets