Ifn γ alexa647

CD-1/ICR mice immunized once with 1 × 10⁷ TCID₅₀ MVA-ZIKV-NS1 vaccine were sacrificed 10 days after immunization, and spleens were harvested. Naïve mouse spleens were harvested in parallel. Isolated splenocytes were analyzed by intracellular cytokine staining following standard protocols. In short, 10⁶ splenocytes were stimulated with 0.1 µg ZIKV NS1 peptide library (JPT Peptide Technology), 0.1 µg HIV Env peptide library (21st Century Biochemicals) or mock stimulated. Incubation proceeded for 6 hours at 37° C +5% CO₂ and GolgiPlug (BD Biosciences) was added at a concentration of 1.0 µL/mL for the last 4 hours of the incubation. The splenocytes were stained with Live/Dead Fixable Green Dead Cell Stain (ThermoFisher) at room temperature for 20 minutes. The samples were treated with Cytofix/Cytoperm (BD Biosciences) at 4° C for 20 minutes and were stained with CD3 APC-CY7, CD4 PE-Cy7, CD8 PerCP, IL2 PE, and IFN-γ Alexa647 (all flow cytometry antibodies from BD Biosciences). Samples were analyzed with a FACSCanto flow cytometer (BD Biosciences) utilizing FACSDiva software. Analysis was performed with FlowJo software. Cytokine responses were scored as a percent of total CD4⁺ (Fig. 4e) or CD8⁺ (Fig. 4f) T cells.

Intracellular staining was performed after incubation with brefeldin A (1 μg/ml, Sigma-Aldrich, Schnelldorf, Germany) for 4 h. Subsequently, the cells were surface stained and fixed of the cells with PBS/ 4% paraformaldehyde. Then cells were permeabilized with 0.5% saponin (Carl Roth, Karlsruhe, Germany) and intracellularly stained with IFN-γ-Alexa647 (BD Bioscience, Pharmingen, Heidelberg, Germany). To determine the expression of the activation marker CD69, the cells were harvested and stained in PBS containing 2% FCS (Thermo Fisher Scientific, Oberhausen, Germany) with CD69-PE-Cy7, CD3-Horizon V450, CD56-PE (BD Pharmingen, Heidelberg, Germany) for 15 min at 4°C in the dark, washed and analyzed via flow cytometry. Degranulation of NK cells was determined by measuring CD107a-PE, CD3-FITC, CD56-APC (BD Pharmingen, Heidelberg, Germany) and Vγ9-Alexa488 (for γδT cell staining (78 (link)) as generous gift from Dietrich Kabelitz and Daniela Wesch). Human PBMCs were co-cultured with tumor cells and CD107a-PE was added for 1 h. Degranulation was blocked with 5 μg/ml Monensin A (Sigma-Aldrich, Schnelldorf, Germany) for an additional 3 h. Flow cytometric analysis was performed using the LSR II from BD Bioscience and data were analyzed with FlowJo® (Tree Star, Switzerland).

Intracellular staining to characterize primary Th2 cells was previously described in [42 , 43 (link)]. Briefly, cytokine staining for IL-4, IL-13 and IFNγ was performed following 4 h of stimulation with PMA (20 ng/mL) and ionomycin (1 μM) in the presence of Brefeldin A (10 μg/mL, all from Sigma Aldrich, Canada). Cells were fixed on ice (10 min) with paraformaldehyde (4%; Sigma Aldrich, Canada) and permeabilized on ice (10 min) with saponin (0.4%, Sigma Aldrich, Canada). Cells were incubated with at 4 °C (30 min) with IL-13-PE (Clone JES10-5A2, isotype rat IgG1κ PE), IFNγ-Alexa 647 (Clone B27, isotype mouse IgG1κ Alexa 647) or IL-4-Alexa 488 (Clone 8D4–8, isotype mouse IgG1κ Alexa 488) antibodies (BD Pharmingen, ON, Canada) and then washed with buffer. Fluorescence was assessed immediately using a FACS Calibur (Becton Dickson, ON, Canada) and data analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA).