Multiparameter Flow Cytometry for T-cell Immune Responses

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.

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Munson P., Liu Y., Bratt D., Fuller J.T., Hu X., Pavlakis G.N., Felber B.K., Mullins J.I, & Fuller D.H. (2018). Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection. Human Vaccines & Immunotherapeutics, 14(7), 1820-1831.

Publication 2018

Live/Dead Yellow CD3-APC CD4-PerCP Cy5.5 CD8 APC-Cy7 IFNγ-BV650 IL-2-PE TNFα-PECy7 CD107a-FITC LSR II flow cytometer

Antibodies Cells Clone Cy5 5 Dmso Fitc Flow cytometry Ifnγ Immune responses Ionomycin Paraformaldehyde Peptide T cell Tnfα Tree

Corresponding Organization :

Other organizations : University of Washington, National Cancer Institute, Center for Cancer Research, Frederick National Laboratory for Cancer Research

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Variable analysis

independent variables

Peptide(s)

dependent variables

T-cell immune responses
Percentage of cells positive for IFNγ, IL-2, TNFα, and CD107a

control variables

Incubation time (10-14 hours)
Number of PBMC (1 million)
Staining panel (Live/Dead Yellow, CD3, CD4, CD8, IFNγ, IL-2, TNFα, CD107a)

controls

Negative control (DMSO)
Positive control (PMA/Ionomycin)

Annotations

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