Fig. 4. Influence of Viola tricolor on IFN-γ and TNF-α production and on degranulation capacity of lymphocytes.
Purified lymphocytes (2 × 105 cells) were incubated for 36 h with cyclosporine A (CsA, 5 μg/mL) or the Viola extract (100 μg/mL), were stimulated with PHA-L (10 μg/mL) and getting a re-stimulation impulse with PMA (50 ng/mL) and ionomycin (500 ng/mL) before analysis of IFN-γ (A) and TNF-α (B) in the supernatant. Activated cells incubated with medium alone were used as controls. Limit of quantification for IFN-γ and TNF-α ELISA was 1.6 and 3.2 pg/mL, respectively. The degranulation capacity was detected by using the LAMP-1 (CD107a) assay and anti-human CD8 mAb staining. Supernatants and cells were analyzed by flow cytometry. Representative dot plots indicate the data (%) from gated living lymphocytes (C). Results are summarized in graphs and data are presented as mean ± SD from three to four independent experiments (D). n.d.: Not detected in the assay. The asterisks represent significant differences of Viola extract-treated cells in comparison to control cells (**P<0.01 and ***P<0.001).