Cytokine detection in supernatants of stimulated PBMC
PBMC (1 × 106
cells) isolated on D40 were stimulated with 20 µg/mL Mhyo antigen or 10
ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma–Aldrich) plus 250 ng/mL ionomycin
(Sigma–Aldrich) (PMA/Iono). Supernatants harvested at 65 h post-stimulation were analyzed
for interleukins (IL) 1β, IL4, IL8, IL10, IL12, and chemokine ligand 2 (CCL2) by
multiplexed fluorescent microsphere immunoassay (FMIA) and IFNγ using an ELISA.