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3 protocols using thy1.2 clone 53 2 1

1

Multiparametric Immune Phenotyping of Cells

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Single cell suspensions were treated with Zombie violet as per the manufacturers insert instructions (Biolegend). Following treatment with Fc block (1 μg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145– 2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4–4), CD4 (clone RM 4–5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11–18H10.1) antibodies (BioLegend). Data was acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the supplementary data.
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2

Influenza Vaccine Formulations and Immunological Analysis

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The following influenza vaccine formulations were purchased from Moore Medical: FluMist Quadrivalent, 2014–2015, live-attenuated vaccine (MedImmune) and Fluzone Quadrivalent, 2014–2015, inactivated virus vaccine (Sanofi Pasteur) directed against A/California/7/2009 (H1N1)pdm09, A/Texas/ 50/2012 (H3N2), B/Massachusetts/2/2012, and B/Brisbane/60/2008; and 2015-2016 FluMist Quadrivalent and Fluzone Quadrivalent formulations directed against A/California/7/2009 (H1N1)pdm09, A/ Switzerland/9715293/2013 (H3N2), B/Phuket/3073/2013, and B/Brisbane/60/2008 strains. Fluorescently conjugated antibodies for Thy1.2 (clone 53-2.1, BioLegend), CD4 (clones RM4-5, BD Biosciences, and GK1.5, eBioscience), CD8 (clone 53-6.7, BioLegend), CD44 (clone IM7, BioLegend), CD62L (clone MEL- 14, BioLegend), CD69 (clone H1.2F3, eBioscience), and CD103 (clone 2E7, eBioscience) were purchased. The influenza NP–specific (NP366–374) H-2Db tetramer was purchased from MBL International. FTY720 was purchased from Cayman Chemical Company.
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3

Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were treated with Zombie violet as per the manufacturer's insert instructions (Biolegend). Following treatment with Fc block (1 µg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145-2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4-4), CD4 (clone RM 4-5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11-18H10.1) antibodies (BioLegend). The data were acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the Supplementary Data.
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