Golgistop protein transport inhibitor

Monocyte-derived DCs and macrophages were counted, washed, and then co-cultured with allogeneic peripheral blood lymphocytes (PBL) for three, five, or nine days in RPMI-1640 medium (Sigma-Aldrich) at a moDC/macrophage: T-cell ratio of 1: 10 at 37 °C. To determine which T-lymphocyte populations were polarized by the preconditioned DCs and macrophages after three, five or nine days the T cells were stimulated with 1 µg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 h, and the vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) 12 h before the cell staining. The cells were labeled with anti-human CD4-Peridinin Chlorophyll Protein Complex (PerCP), CD8-PE and/or anti-human CD25-PE-conjugated antibodies (BioLegend). Following this, they were fixed and permeabilized by using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend), and anti-human FoxP3-APC (R&D System) antibodies. Fluorescence intensities were measured by FACS Calibur cytometer (BD Biosciences) and data were analyzed by the FlowJo v X.0.7 software (Tree Star).

The lymph nodes (pool of inguinal and mesenteric) were collected on the 7^th day after immunization. The cells were isolated by flushing the tissue through a cell strainer (70 μm) with RPMI medium, followed by centrifugation at 450 g for 5 min at 4 °C and the pellet was resuspended in 3 ml of RPMI medium. In a 24-well plate, the cells were cultured at a concentration of 1 × 10⁶ cells in 500 μl in RPMI medium with or without MOG_35–55 (1 µg/ml) and BD GolgiStop™ Protein Transport Inhibitor (provided in the kit or Cat #554724) for 12 h. After 8 h, cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4 h. The cells were stained for surface markers and intracellular cytokine with Mouse Th17/Treg Phenotyping kit (#560767, BD Biosciences) according to the manufacturer’s directions. Samples were analyzed using a FACS Canto II Flow Cytometer (BD Biosciences, San Diego, USA) after the acquisition. Data analysis was performed using FlowJo VX.0.7r2 (Tree Star, Ashland, OR). The strategy for analysis and representative dot plots of the experiments are shown in the Supplementary Material.

For NK cell activation phenotype, NK cells were stained with a cocktail of directly conjugated antibodies against surface molecules, including anti-CD56 (APC), -CD3e (FITC), -CD54 (PE), -CD69 (PE), -NKp46 (PE), and -CD158b (PE) and followed by flow cytometry. For NK cell function, NK cells were cocultured with K562 cells at 1:1 effector–target (E/T) ratio for 1 h and followed by intracellular staining for CD107a, granzyme B and perforin and followed by flow cytometry. For IFN-γ production, NK cells were cocultured with K562 cells for 2 h, thereafter, BD GolgiStop™ Protein Transport Inhibitor (contains monensin) was added to the coculture and incubation for another 4 h. The cells were then stained intracellularly with anti-IFN-γ antibody and followed by flow cytometry. In some experiments, NK cells were pretreated with specific inhibitor of SHIP-1 (3AC) (33 (link)) (0.5 µM), ERK (U0126) (10 µM), and JNK (SP600125) (25 µM), or vehicle controls (culture medium containing 0.002% ethanol for 3AC, and 0.1% DMSO for both U0126 and SP600125) for 16 h. All the experiments were performed using FACS Calibur (Becton Dickinson). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Mean fluorescence intensity (MFI) and percentage positive cells were determined.

Lymphocytes were isolated from spleens after organ removal and analyzed separately for each mouse by flow cytometry. Cell surface stainings were performed according to standard procedures using antibodies against CD4, CD8, CD19 and CD25 directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) purchased from BD Pharmingen (San Diego, CA). Anti-CD4-Tri-Color was purchased from Caltag Laboratories (Hamburg, GER). Monoclonal antibodies (mAbs) for intracellular stainings (anti-IL-4⁺, anti-IL-17⁺, anti-IFNγ⁺) conjugated to PE or allophycocyanin (APC) were purchased from BD Pharmingen. PE- or FITC-labelled mAbs to FoxP3 were from eBioscience (Vienna, AUT). Intracellular cytokine staining was performed with PE- or APC- labelled mAbs as described [41 (link)]. In brief, isolated cells were stimulated with plate-bound anti-mouse CD3e mAbs (clone 145-2C11), and anti-mouse CD28 mAbs (clone 37.51, both BD Pharmingen) at 1μg/ml for 5 hours under the presence of BD-GolgiStop Protein Transport Inhibitor (containing Monensin, BD Biosciences, San Diego, CA). All flow cytometry was performed on a FACSCanto II and analyzed using FACSDiva software (all from BD Biosciences). The main cell types were quantified as their percentage in the entire lymphocyte populations, while T-cell subsets where quantified as their percentage in CD4+ lymphocyte populations.

Mouse splenocytes were cultured in 12-well round bottom plates (4×10⁶ cells/well) and stimulated with or without mixed T. gondii peptides (4 μg/ml/peptide) for 24 h. Cells were treated with BD GolgiStop™ protein transport inhibitor (BD Biosciences, San Diego, USA) for 6 h post stimulation. Then, cells were recovered and blocked with anti-mouse CD16/32 (Biolegend, CA, USA). After washing twice with cell staining buffer (BD Biosciences), cells were prepared in 50μl staining buffer and stained with FITC anti-mouse CD3ε (Biolegend, CA, USA) and with APC anti-mouse CD8α (Biolegend, CA, USA) at 4°C for 30 min. Following two additional washes, cells were thoroughly suspended in 250 μl fixation/permeabilization solution (BD Biosciences) for 20 min at 4°C. Then cells were washed, re-suspended in 50μl of BD Perm/Wash solution and stained with PerCP/Cy5.5 anti-mouse IFN-γ (Biolegend, CA, USA) at 4°C for 30 min. Finally, IFN-γ expression in CD8⁺CD3⁺ T cells was measured by BD Accuri™ C6 flow cytometer.