Monensin golgi stop

Splenocytes isolated from randomly selected mice in different groups were isolated as described above and 2X10⁶ splenocytes per well were cultured in RPMI 1640 (Gibco, Invitrogen, UK) containing 10% FBS in a 12-well plate (Nunc, USA) at 37^°C in 5% CO₂ for 4 hours to bring the cellular activity to basal levels. Splenocytes were then challenged with 20 μg/ml of H37Rv CSA and cultured for an additional 2 hours, after which 5 μl Monensin (Golgi-Stop, BD Biosciences) and 5 μl of anti-CD107A-FITC antibody (BD Biosciences) were added per well and cultured for an additional 4 hours at 37°C in 5% CO₂. These cells were then collected and surface-stained for FACS analysis.

Lymph node cells were stimulated with 2 µg/ml H56 together with anti-CD28 (37.51; BD Biosciences) and anti-CD49d (9C10; BD Biosciences) for 1 h. Subsequently, 10 µg/well brefeldin A (Sigma-Aldrich, St. Louis, MO) and 0.7 µl/well monensin/GolgiStop (BD Biosciences Franklin Lakes, NJ) were added and cells were incubated for 5 h at 37 °C. After overnight storage at 4 °C, cells were washed in FACS buffer (1% FCS (VWR-Bie & Berntsen, Herlev, Denmark), 0.1% sodium azide (VWR, Radnor, PA) in PBS (Life Technologies, Carlsbad, CA), and stained for surface markers with 1 µg/ml anti-CD4-APC-eFlour780 (clone GK1.5) and 1 µg/ml anti-CD44-FITC (clone IM7) (both eBiosciences, San Diego, CA) for 30 min at 4 °C. Cells were washed with FACS buffer, before fixation and permeabilization using Cytofix/Cytoperm kit (BD Biosciences Franklin Lakes, NJ). Subsequently, cells were stained for intracellular cytokines with 1 µg/ml anti-IFN-γ PE-Cy7 (XMG1.2; eBiosciences, San Diego, CA), 1 µg/ml anti-TNF-PE (MP6-XT22; eBiosciences, San Diego, CA) and 1 µg/ml IL-17A for 20 min. Finally, cells were washed, re-suspended in FACS buffer, and analyzed using a FACSCanto flow cytometer (BD Biosciences Franklin Lakes, NJ) and FlowJo software version 10.

To assess viability, cells were stained with LIVE/DEAD fixable NEAR-IR dead cell stain kit according to manufacturer’s protocol (Life Technologies). To stain CD107a, cell cultures were supplemented with CD107a/FITC mAb (or control IgG₁/FITC) for 6 h, with the addition of monensin (Golgi stop, diluted 1:1500; BD Pharmingen) and 10 μg/ml Brefeldin A (Sigma-Aldrich) after the first hour. The cell-surface and intracellular staining procedures were performed as described previously^{14 (link)}. Immunolabeled cells resuspended in 1% paraformaldehyde (PFA) were acquired on a FACSCanto II (BD Biosciences) or Cytoflex (Beckman Coulter). Positive cell gating was set using fluorescence minus one control (FMO). Mean fluorescence intensity (MFI) was subtracted of the value obtained with isotype control antibody. Data analyses were performed using FlowJo software (TreeStar).

Single‐cell suspensions from spleen, mesenteric lymph nodes and liver were isolated in phosphate‐buffered saline (PBS) containing 1% foetal bovine serum as described previously.12 For CXCL12⁺ Tfh cell analysis, cells were stimulated for 6 hours in culture medium containing phorbol myristate acetate (PMA, 25 ng/mL; Sigma‐Aldrich), ionomycin (1 μg/mL; Sigma‐Aldrich) and monensin (Golgi Stop; 1 μg/mL; BD Biosciences). Cells then were incubated for 30 minutes at 4°C with the following monoclonal antibodies: CD3e‐Percp‐cy5.5 (clone 145‐2C11, eBioscience), CD4‐PE‐Cy7 (clone GK1.5, eBioscience), PD‐1‐PE (clone J42, eBioscience) and CXCR5‐FITC (clone 2G8, BD Pharmingen). After staining of surface markers, the cells were permeabilized with cold Fix/Perm Buffer, and incubated with CXCL12‐APC (clone 79018, R&D Systems) after blockade with antimouse CD16/32 (clone 93, eBioscience). For eosinophil surface marker analysis, cells were incubated with Siglec‐F‐PE (clone E50‐2440, BD Pharmingen), CXCR4‐FITC (clone 2B11/CXCR4, BD Pharmingen) or isotype (BD Pharmingen) antibodies. Cells acquisition was performed using a FACSVerse cytometer (Lasers: 488 and 633; Mirrors: 507 LP, 560 LP, 665 LP, 752 LP, 660/10, and 752 LP; Filters: 488/15, 527/32, 568/42, 700/54, 783/56, 660/10 and 783/56, BD Biosciences). Data were analysed with FlowJo (Tree Star, version 10.0.7).