Monensin

Magnetic enrichment and phenotypic analysis of porcine antigen-reactive CD4+ T was performed as previously described (42 (link)). In brief, adult and L3 A. suum lysate (40µg/ml) was added to 3×10⁷ PBMC or lung tissue-derived lymphocytes, and cells were cultured in the presence of the anti-human mAb CD40L-PE (1:50 dilution, clone 5C8, Miltenyi Biotec) at 37°C, 5% CO₂ for 5h. Unstimulated cells (w/o) served as control. CD40L expression on the surface was stabilized by adding Monensin (2 µM, eBiosciences) after 2h of stimulation. After 5h of stimulation, CD40L-expressing cells were magnetically labeled using anti-PE-microbeads (Miltenyi Biotec) and further cultured in the presence of Brefeldin A (1 µg/ml, eBiosciences) and Monensin (1 µg/ml, eBioscience) to allow for the detection of intracellular cytokine expression. Finally, cells were magnetically enriched and stained on two sequential MS columns (Miltenyi Biotec) using the Inside stain kit (Miltenyi Biotec) before being acquired on a BD FACS Aria III.

Splenocytes were stained for viability and surface markers CD3, CD4, CD8, CD44, and CD62L. Intracellular staining for FoxP3 was performed after surface marker staining followed by fixation and permeabilization (eBioscience). For intracellular cytokine staining, cells were resuspended in 10% heat inactivated FBS-RPMI medium containing 1× Monensin (Invitrogen) and cultured at 37°C and 5% CO₂ for 4 h. After viability and surface marker staining, cell fixation and permeabilization was performed followed by intracellular staining for IL-10 and IFN-γ for flow cytometry.