Multicolor Flow Cytometry Phenotyping

After co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 14-color panel containing Yellow Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA) and mAb to CD14-BV570 (M5E2, Biolegend), CD19-BV570 (HIB19, Biolegend), CD3-BV650 (OKT3, Biolegend), CD4-PECy5 (RPA-T4, BD), CD8-PerCP-Cy5.5 (SK1, BD), CD45RA-biotin (HI100, BD), CD62L-APC-A780 (DREG-56, Ebioscience), integrin α₄β₇-A647 (ACT1; conjugated in house) streptavidin(SAV)-Qdot800 (Invitrogen) CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), TNF-α-A700 (MAb11, BD), IL-2-BV605 (MQ1-17H12, Biolegend), IL-17A-BV421 (BL168, Biolegend), and MIP-1β-PE (IC271P, R&D) as previously described (11 (link)). Samples were acquired using a customized LSRII flow cytometer (BD Biosciences) and analyzed using Winlist v7.0 (Verity Software House, Topsham, ME, USA). Absolute numbers of CD3, CD8, CD4 positive cells and CD8 memory subsets cells were calculated by using percentages obtained from flow cytometry analysis related to the absolute number of lymphocytes determined by routine blood count. Responses against S. Typhi were expressed as net percentage of positive cells (i.e., total percentage of positive cells in the presence of S. Typhi-infected targets minus percentage of positive cells in co-cultures with uninfected cells).

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Fresnay S., McArthur M.A., Magder L.S., Darton T.C., Jones C., Waddington C.S., Blohmke C.J., Angus B., Levine M.M., Pollard A.J, & Sztein M.B. (2017). Importance of Salmonella Typhi-Responsive CD8+ T Cell Immunity in a Human Typhoid Fever Challenge Model. Frontiers in Immunology, 8, 208.

Publication 2017

CD14-BV570 (M5E2, CD3-BV650 (OKT3, CD4-PECy5 (RPA-T4, IFN-γ-PE-Cy7 IL-17A-BV421 LSRII flow cytometer

Biotin Blood Cd4 cells Cd62l Cd8 cells Cells Cells cultures Cy5 5 Flow cytometry Ifn γ Il 17a Integrin Lymphocytes number Memory Okt3 Streptavidin Tnf α

Corresponding Organization : University of Maryland, Baltimore

Other organizations : Oxford BioMedica (United Kingdom), University of Oxford

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Variable analysis

independent variables

Stimulator cells co-culture

dependent variables

Percentage of positive cells for the following markers: CD3, CD8, CD4, CD8 memory subsets, IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β
Absolute numbers of CD3, CD8, CD4 positive cells and CD8 memory subsets cells

control variables

PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly
14-color panel containing Yellow Live/Dead viability kit, CD14-BV570, CD19-BV570, CD3-BV650, CD4-PECy5, CD8-PerCP-Cy5.5, CD45RA-biotin, CD62L-APC-A780, integrin α4β7-A647, streptavidin(SAV)-Qdot800, CD69-ECD, IFN-γ-PE-Cy7, TNF-α-A700, IL-2-BV605, IL-17A-BV421, and MIP-1β-PE

controls

Uninfected cells as a negative control

Annotations

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