Multiparameter flow cytometry was used to determine T-cell immune responses using peptide stimulated PBMC as previously described.23 (link) One million PBMC were stained for each condition (DMSO, PMA/Ionomycin, or peptide(s)) for 10–14 hours. PBMC were stained with the following antibodies: Live/Dead Yellow (Invitrogen®, Catalog #:L34959), CD3-APC (BD Biosciences, clone SP34-2, Catalog #: 551916), CD4-PerCP Cy5.5 (BD BioSciences, clone L200, Catalog #: 551980), CD8 APC-Cy7 (BD Biosciences, clone RPA-T8, Catalog #: 55760), IFNγ-BV650 (BioLegend, clone 4S.B3, Catalog #: 502537), IL-2-PE (BioLegend, clone MQ1-17H12, Catalog #: 500307), TNFα-PECy7 (BD Biosciences, clone Mab11, Catalog #: 557647), CD107a-FITC (BD Biosciences, clone H4A3, Catalog #: 555800). Cells were fixed in 1% paraformaldehyde and acquired using an LSR II flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (Tree Star, Inc.). Samples were considered positive if peptide-specific responses were at least twice that of the negative control plus at least 0.01% after background subtraction.
Ifnγ bv650
Manufactured by BioLegend
IFNγ-BV650 is a fluorochrome-conjugated antibody designed for the detection and quantification of interferon gamma (IFNγ) in flow cytometry applications. The BV650 fluorochrome provides a bright and stable signal for sensitive IFNγ detection.
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2 protocols using ifnγ bv650
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Multiparameter Flow Cytometry for T-cell Immune Responses
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Multiparameter Flow Cytometry of T-cell Responses
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Multiparameter flow cytometry to determine T-cell immune responses was performed using peptide (GFP or AAV9 peptide pools)- stimulated PBMC as previously described (50). One million PBMC were stained for each condition (DMSO, PMA/Ionomycin, and peptide(s)) for 10–14 hours. PBMC were stained with the following antibodies Live/Dead Yellow (Invitrogen®), CD3-APC (BD Biosciences, clone SP34-2), CD4-PerCP Cy5.5 (BD BioSciences, clone L200), CD8 APC-Cy7 (BD, clone RPA-T8), IFNγ-BV650 (BioLegend, clone 4S.B3), IL-2-PE (BioLegend, clone MQ1-17H12), TNFα-PECy7 (BD, clone Mab11), CD107a-FITC (BD, clone H4A3). Cells were fixed in 1% paraformaldehyde and acquired using an LSR II flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR). Samples were considered positive if peptide-specific responses were at least twice that of the negative control plus at least 0.01% after background subtraction.
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