Golgistop

Then, 2 weeks after the last immunization, the spleen homogenates of immunized mice were filtrated through a cell strainer (40 µm; BD Biosciences) in RPMI 1640 medium (GIBCO) containing 10% fetal bovine serum (GIBCO) and then red blood cells (RBCs) were lysed with RBC lysis buffer (Sigma-Aldrich) for 3 min at room temperature. Single-cell suspension (2 × 10⁶ cells) was incubated with 10 μg/mL VLP_FMDV, 2 μg/mL anti-CD28 monoclonal antibody (clone 37.51; eBioscience, San Diego, CA, USA), 0.5 μg/mL GolgiStop (eBioscience), and 0.5 μg/mL GolgiPlug (BD Biosciences) for 12 h at 37 °C. Next, the cells were washed with cold PBS and stained with live/dead staining kit (InvivoGen, San Diego, CA, USA), anti-CD4-BV450 (clone RM4-5; BD Biosciences), anti-CD8α-FITC antibodies (clone 53-6.7; BD Biosciences), anti-CD3e-APC-Cy7 (clone UCHT1; eBioscience) at 4 °C for 30 min. The cells were fixed using Cytofix/Cytoperm Plus Kit (BD Biosciences), washed, and then stained intracellularly with anti-mouse IFN-γ-PE (anti-mIFN-γ-PE; clone XMG1.2; BD Bioscience), anti-mIL-5-APC (clone TRFK5; BD Bioscience), and anti-mIL-17A-PE-Cy7 (clone eBio17B7; eBioscience) at 4 °C for 30 min. The stained cells were analyzed with MACSQuant VYB flow cytometer (Milteny Biotech, San Diego, CA, USA) and the results were analyzed with FlowJo software (TreeStar, Ashland, OR, USA).

To create single-cell suspensions, harvested spleens and lungs were incubated in RPMI 1640 digestion media (10% fetal bovine serum, 0.1% collagenase type II (Worthington), 1 mM MgCl₂, and 1 mM CaCl₂) at 37°C for 30 min. The single-cell suspensions were then filtered through a 40-μm cell nylon mesh cell strainer, treated with RBC lysis buffer (Sigma) for 5 min, and washed twice with RPMI 1640 medium supplemented with 2% FBS. Single-cell suspensions from the spleens and the lungs of immunized mice were stimulated with Rv3628 (5 μg/ml) for 12 h at 37°C in the presence of GolgiStop (eBioscience). The cells were first blocked with Fc Block (anti-CD16/32; eBioscience) for 15 min at 4°C and then stained with fluorochrome-conjugated antibodies against CD4, CD8, CD62L, CD44, CD127 (eBioscience) and CD3 (BD bioscience) for 30 min at 4°C. Cells stained with appropriate isotype-matched immunoglobulins were used as negative controls. The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Intracellular TNF-α, IL-2, T-bet, GATA-3, RORγt (eBioscience) and IFN-γ (BD bioscience) levels were detected with fluorescein-conjugated antibodies in a permeation buffer. The cells were analyzed with a FACSverse flow cytometer using FlowJo software.

Mononuclear cells were stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’ (eBioscience, San Diego, CA) as described previously [33 (link)]. Briefly, cells were stained with surface antibodies CD8 Amcyan (BD Biosciences. Oxford, UK), CD3 APC-Cy7 (Cambridge Biosciences, Cambridge, UK), CCR4 PE-Cy7 (BD Biosciences, Oxford, UK) and CXCR3 PE (BD Biosciences. Oxford, UK) for 20 mins at 4°C. Samples were washed, re-suspended in RPMI with 10% human serum (TCS Biosciences, Buckingham, UK) and then stimulated with PMA and ionomycin for six hours in the presence of ‘Golgi stop’. Cells were washed, stained with a dead cell exclusion dye (Invitrogen, Oregon, USA) at 4°C for 20 mins and then washed again before fixation with 2% paraformaldehyde at room temperature for 10–15 mins. After further washing, cells were permeabilised with 0.5% saponin for 5 minutes followed by incubation with the intracellular antibodies IL-17A (BioLegend, London, UK) (2.5 ml), IFN-γ (BioLegend, London, UK) (0.5 ml) and isotype controls for 30 mins at room temperature. Samples were washed and analysed by flow cytometry using on LSR II flow cytometry. To define Th17 cells we measured the secretion of IL-17 using intracellular staining and FACS analysis.

Three weeks after the final immunization of antigens, single-cell suspensions (1 × 10⁶/mL) from the lung and spleen of adjuvant-, BCG-, and antigen-immunized mice were stimulated with purified proteins (InsB, 2 μg/mL; ESAT-6, 2 μg/mL) for 24 h at 37°C. IFN-γ cytokine levels were analyzed in the culture supernatant via sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol. Additionally, single-cell suspensions were stimulated with purified proteins (InsB; 2 μg/mL, ESAT-6; 2 μg/mL) for 12 h at 37°C in the presence of GolgiStop (eBioscience, San Diego, CA, United States), and then the cells were stained with Live/Dead Stain (InvivoGen, San Diego, CA, United States) and with anti-CD4 (PerCp-Cy5.5, eBioscience), anti-CD8 (APC-Cy7, eBioscience), and anti-CD3 (BV421, eBioscience) antibodies for 30 min at 4°C. Next, the stained cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s protocol and then stained with anti-IFN-γ (PE, eBioscience). The cells were analyzed with a FACSVerse flow cytometer using FlowJo software.