Antigenic stimulation of PBMC for proliferation and cytokine secretion was performed according to standard procedures (Mustafa 2009b). In brief, 2 × 105 PBMC suspended in 50 μL complete medium was seeded into the wells of 96-well tissue culture plates (Nunc, Roskilde, Denmark). Antigens
in 50 μL complete medium were added at optimal concentrations to the wells in triplicates. Whole bacilli were used at 10 μg mL−1 (wet weight) and all other antigens and peptides were used at an optimal concentration of 5 μg mL−1. The cells in the control wells did not receive any mycobacterial antigen/peptide. The final volume of the culture in each well was adjusted to 200 μL. Con A 10 μg mL−1 (Sigma Chemical,
St. Louis, MO) was used as a positive control. The plates were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. On day 6, culture Bortezomib manufacturer supernatants (100 μL) were collected from each well and frozen at −20 °C until used to determine cytokine concentrations. The remaining cultures were pulsed with 1 μCi 3H-thymidine (Amersham Life Science, Amersham, UK) and harvested (Skatron Instruments AS, Oslo, Norway) according to standard procedures (Al-Attiyah et al., 2003). The incorporated radioactivity was obtained as counts per minute (c.p.m.). see more The average c.p.m. was calculated from triplicate cultures stimulated with each antigen or peptide pool, as well as from triplicate wells of negative control cultures lacking antigen. The cell proliferation results were presented as stimulation index (SI), where SI is the c.p.m. in antigen- or peptide-stimulated Dolichyl-phosphate-mannose-protein mannosyltransferase cultures per c.p.m. in cultures lacking antigen or peptide. A patient was considered to be a responder to a given antigen if the PBMC yielded SI≥3 (Al-Attiyah et al., 2003). Positive responses ≥60% were considered strong, 40% to <60% moderate, and
<40% weak (Mustafa, 2009a, b). The supernatants, collected from the cultures of PBMC of TB patients (n=20) and healthy subjects (n=12) before 3H-thymidine pulse, were randomly selected for assays to determine concentrations of secreted IFN-γ and IL-10 using FlowCytomix kits (Bender Medsystems GmbH, Vienna, Austria), according to the manufacturer’s instructions (Al-Attiyah & Mustafa, 2008, 2009). These kits allow simultaneous quantification of cytokines including IFN-γ and IL-10. In brief, FlowCytomix technology is based on spectrally discrete microspheres that are used as solid phase in an immunoassay. The beads are internally dyed with Starfire Red, a far red (685–690 nm) emitting fluorochrome, which is excited by UV, argon or HeNe lasers. The test samples were analyzed by flow cytometry using Coulter EPICS FC500 (Beckman Coulter Inc., USA). For each analysis, up to 10 000 events were acquired. The mean concentration of each cytokine was expressed as pg mL−1.
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