Multiparametric Flow Cytometry Analysis

Following overnight co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 13-color panel as previously described [24 (link), 30 (link), 31 (link)]. The panel used included the following mAbs: CCR7-PE (clone: G043H7, provider: BioLegend), CD4-PerCP-Cy5.5 (L200, BD Biosciences), CD19-BV421 (HIB19, Biolegend), CD3-BV650 (SP34-2, BD Biosciences), integrin α4β7-Alexa Fluor 647 (ACT1, conjugated in house), CD8-Alexa Fluor 700 (RPA-T8, BD Biosciences), CD45RA-APC-Cy7 (5H9, BD Biosciences), CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), IL-17A-BV570 (BL168, Biolegend), IL-2-BV605 (MQ1-17H12, BD Biosciences), and TNF-α-BV711 (MAb11, Biolegend). Cell viability was assessed using a Violet Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA). Stained cells were fixed with 1% PFA in PBS. Samples were acquired using a customized LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo v10 (FlowJo, LLC, San Francisco, CA). Responses against IpaB (S. flexneri 2a) were expressed as net percentage of positive cells (i.e., total percentage of positive cells in the presence of IpaB-stimulating cells (targets) minus percentage of positive cells in co-cultures with background stimulators (B-LCL without IpaB).

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Toapanta F.R., Bernal P.J., Kotloff K.L., Levine M.M, & Sztein M.B. (2018). T cell mediated immunity induced by the live-attenuated Shigella flexneri 2a vaccine candidate CVD 1208S in humans. Journal of Translational Medicine, 16, 61.

Publication 2018

CCR7-PE CD4-PerCP-Cy5.5 CD19-BV421 (HIB19, CD8-Alexa Fluor 700 (RPA-T8, CD69-ECD IFN-γ-PE-Cy7 LSRII flow cytometer

Alexa fluor 647 Cell viability Cells Cells culture Clone Co cultures Cy5 5 Ifn γ Il 17a Integrin Mabs Tnf α Violet

Corresponding Organization :

Other organizations : University of Maryland, Baltimore

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Variable analysis

independent variables

Stimulator cells (used for overnight co-culture with PBMC)

dependent variables

Percentage of positive cells expressing cytokines (IFN-γ, IL-17A, IL-2, TNF-α) in response to IpaB (S. flexneri 2a) stimulation

control variables

Overnight co-culture conditions (temperature, duration, etc.)
PBMC harvesting and washing protocol
Staining and permeabilization procedures
Flow cytometry panel (13-color) and settings
Gating and data analysis using Flowjo v10

positive controls

Co-cultures with background stimulators (B-LCL without IpaB)

negative controls

None specified

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