Cell countess 3

Cell stimulation was done by incubating for 48 h 5 × 10⁶ Jurkat or macrophage-derived THP-1 (resting M0) cells with basal medium, or medium samples collected from the basal side of VLC cultures at day 6–7 post-infection. Note that cell viability was assessed by trypan blue counterstaining using Cell countess 3 (Invitrogen) and was above 80% at the time of stimulation. Positive controls of cell activation were also included and generated as following. Jurkat cells activation was done by 48 h of phytohemagglutinin (Sigma, 10 μg/ml) and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation. Differentiation of THP-1 into resting macrophage (M0) was done by incubation with phorbol 12-myristate 13-acetate (185 ng/ml, Sigma–Aldrich) for 24 h. Polarization of M0 into a macrophage with a pro-inflammatory phenotype (M1 positive control) was done by 48 h of INF-γ (20 ng/ml, Immunotools), LPS (100 ng/ml, Sigma–Aldrich), and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation. Polarization of M0 into a macrophage with an anti-inflammatory phenotype (M2a positive control) was done by 48 h of IL-4 (20 ng/ml, Immunotools), IL-13 (20 ng/ml, Immunotools), and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation.