Cytometric bead array multiplex assay

PLF and plasma levels of interleukin- (IL-) 10, tumor necrosis factor- (TNF-) α, interferon- (IFN-) γ, and monocyte chemotactic protein-1 (MCP-1) were measured by cytometric bead array multiplex assay (BD Biosciences, San José, CA, USA) according to the manufacturer's recommendations. IL-1β, IL-6, and cytokine-induced neutrophil chemoattractant (KC, CXCL1) were measured by ELISA (R&D Systems, Abingdon, UK) in accordance with the manufacturer's instructions. Lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT), alanine transaminase (ALAT), creatinine, and urea levels were measured in plasma using kits from Sigma (St. Louis, MO, USA) by a Hitachi analyzer. Nucleosome levels were determined in PLF by ELISA as described previously [22 (link)].

Plasma samples were stored immediately at −20°C after obtention. Soluble granzyme A and B were measured by sandwich ELISA (eBioscience; LD 2 pg/ml) in plasma, in accordance with the manufacturer’s recommendations. Human tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, and IFN-γ were measured by cytometric-bead-array multiplex assay (BD Biosciences; LD 0.5 pg/mL). Aspartate transaminase (AST), alanine transaminase (ALT) and renal function were measured in plasma with spectrophotometry (Roche Diagnostics) as for previous study [16 (link)].

Unless stated otherwise, purified monocytes at a concentration 2.5 × 10⁵ cells/ml in RPMI 1640 culture medium were pretreated with 50 μM glibenclamide (Sigma) [comparable to the peak human plasma concentration achieved following a 20 mg oral dose (23 (link))] for 30 min and then infected with live Mtb or BCG at 10² or 10⁵ CFU per well or activated with 10 μg/ml of LPS (from Escherichia coli, Sigma) at 37°C for 96 h. The supernatants were stored at −80°C until cytokines were measured. TNF-α, IL-10, and IL-6 concentrations were tested in duplicate by ELISA (Invitrogen and BD Biosciences) according to the manufacturer's instructions. IL-1β concentration was measured using Quantikine HS ELISA (R and D system). IL-8, MCP-1, RANTES, IP-10, and MIG were determined using a cytometric bead array multiplex assay (CBA) in accordance with the manufacturer's instructions (BD Biosciences). All cytokine data in response to Mtb, BCG, or LPS were subtracted from the medium control of each sample.

Lung (and cell supernatant) levels of IL-1β, TNF-α, IL-6, IL-10, CXCL-1 and CXCL-2 were measured by ELISA (R&D Systems, Minneapolis, MN). Plasma levels of TNF-α, IL-6, and IL-10 were measured by using a cytometric bead array multiplex assay (BD Biosciences). MPO was measured by ELISA from HyCult Biotechnology (Uden, the Netherlands).Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST were measured using kits from Sigma and a Hittachi analyzer (Boehringer Mannheim).