Monensin 1x

To analyze the magnitude and phenotype of the HIV-1-, FLAG- or VACV-specific T cell immune responses, 2 × 10⁶ splenocytes (erythrocyte-depleted) seeded on 96-well plates were stimulated for 6 h in complete Roswell Park Memorial Institute (RPMI) 1640 medium (100 units/mL of penicillin/100 μg/mL of streptomycin, 2 mM L-glutamine, 10 mM Hepes and 0.01 mM β-mercaptoethanol) with 10% FCS, anti-CD107a-FITC (BD Biosciences), 1 µL/mL Golgiplug (BD Biosciences), monensin 1X (Invitrogen) and 5 µg/mL of the different HIV-1 clade B consensus peptide pools or 5 µg/mL of FLAG peptide or 10 µg/mL of VACV E3 peptide. After stimulation, splenocytes from immunized mice were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and stained intracellularly with the following fluorochrome-conjugated antibodies: IL-2-APC, IFN-γ-PeCy7 and TNF-α-PE for functional analyses and CD3-PECF594, CD4-APCCy7, CD8-V500, CD127-PerCPCy5.5 and CD62L-Alexa700 for phenotypic analyses (all from BD Biosciences). The dead cells were excluded using the violet LIVE/DEAD stain kit (Invitrogen).

To determine the magnitude and phenotype of the SARS-CoV-2-specific T cell responses, 4 × 10⁶ splenocytes or 2 × 10⁶ lung-derived lymphocytes (both cell types were erythrocyte-depleted) seeded on 96-well plates were stimulated ex vivo for 6 h in complete RPMI 1640 medium with 10% FCS, 1 µL/mL Golgiplug (BD Biosciences), anti-CD107a-FITC (BD Biosciences), monensin 1X (Invitrogen) and 1 µg/mL of the different SARS-CoV-2 peptide pools representing the S, M and N antigens (JPT Peptide Technologies GmbH, Berlin, Germany). After stimulation, lymphocytes were stained for surface markers, fixed/permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and intracellularly stained by incubation with the following fluorochrome-conjugated antibodies: IFN-γ-PeCy7, IL-2-APC and TNF-α-PE for functional analyses and CD3-PE-CF594, CD4-APC-Cy7 and CD8-V500 for phenotypic analyses (all from BD Biosciences). Dead cells were excluded from the analysis using the LIVE/DEAD Fixable Violet Dead Cell Stain kit (Invitrogen). Cells were acquired in a GALLIOS flow cytometer (Beckman Coulter), and data analyses were performed using FlowJo software (Version 10.4.2; Tree Star). Lymphocyte-gated events ranged between 10⁵ and 5 × 10⁵. Background responses obtained in unstimulated controls (RPMI) were subtracted from the responses detected in stimulated samples.