Fast immune brefeldin a solution

Whole blood cell cultures were performed to determine the frequencies of intracellular cytokine-producing cells. Briefly, whole blood was diluted 1:1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with SsAg, NIE, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d /CD28 (BD Biosciences) at 37°C for 6 or 18 h, for intracellular cytokine staining or ELISA respectively. Fast Immune Brefeldin A Solution (10μg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1x FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at -80°C until use. For cytokine neutralization experiments (n = 15), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5μg/ml) or anti-TGFβ (5μg/ml) or isotype control antibody (5μg/ml) (R& D Sytems) for 1 h following which NIE and brefeldin A was added and cultured for a further 23 h.

Whole blood cell cultures were performed to determine the F_o of intracellular cytokine-producing cells.. Briefly, whole blood was diluted 1∶1 with RPMI-1640 medium, supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM) (all from Invitrogen, San Diego, CA) and placed in 12-well tissue culture plates (Costar, Corning Inc., NY, USA). The cultures were then stimulated with BmA, Mf, PPD, PMA/ionomycin (P/I) or media alone in the presence of the co-stimulatory reagent, CD49d/CD28 (BD Biosciences) at 37°C for 6 hrs. FastImmune Brefeldin A Solution (10 µg/ml) (BD Biosciences) was added after 2 hours. After 6 hours, whole blood was centrifuged, washed using cold PBS, and then 1× FACS lysing solution (BD Biosciences, San Diego, CA, USA) was added. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Diego, CA, USA), cryopreserved, and stored at −80°C until use. The same procedure was used for both prospectively collected as well as for retrospectively stored samples. For cytokine neutralization experiments (n = 7), whole blood from INF individuals was cultured in the presence of anti-IL-10 (5 µg/ml) or anti-TGFβ (5 µg/ml) or isotype control antibody (5 µg/ml) (R& D Sytems) for 1 h following which BmA and brefeldin A was added and cultured for a further 5 h.