Liberase cl

Single-cell suspensions from BT-II or OT-II mouse lymph nodes were incubated with anti-CD4 MicroBeads isolated on a MACS LS column (Miltenyi Biotec, Singapore). Splenic dendritic cells (DCs) were digested with Liberase Cl (Roche, Switzerland) at 37°C for 30 min and isolated by centrifugation over OptiPrep Density Gradient Medium (Sigma-Aldrich, St Louis, MO), incubated with anti-mouse CD11c MicroBeads, and passed through a MACS LS column (Miltenyi Biotec). BT-II and OT-II CD4 T cells and splenic DCs were cocultured in 48-well plates with 10 μg/ml Blo t 5_55–70 peptide (IIRELDVVCAMIEGAQ) or OVA_323–339, respectively (AnaSpec, Fremont, CA); 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ)1; 20 μg/ml anti-mouse IFN-γ; 20 μg/ml anti-mouse IL-12/IL-23 p40 (eBioscience, ImmunoCell, Singapore); and 20 μg/ml mouse IFN-γ R1/CD119 Ab (R&D Systems, Minneapolis, MN). Cells were restimulated on days 3, 7, and 11 with Blo t 5_55–70, cytokines, and neutralizing Abs. Fresh DCs were added on day 7. Th2-polarized BT-II cells were harvested on day 14.

Perfused lungs from infected mice were cut into 1–2 mm pieces and subsequently digested with liberase Cl (0.4 mg ml⁻¹ in PBS; Roche-Diagnostics, Indianapolis, IN). The reaction was stopped after 30–45 min. with an equal volume of fetal calf serum. Digested lung was fully dispersed by passage through a 100-µm pore-size cell strainer and an aliquot removed for bacterial load measurements. Isolated cells were then washed, counted and subsequently surface stained for FACS sorting in a BSL3 containment facility under sterile conditions. In some experiments FACS-sorted cells or whole-lung single-cell suspensions from infected mice were stimulated overnight in the presence or absence of live Mtb H37Rv (m.o.i. = 1) at 10⁶ cells per well in 96-well plates. Supernatants were then examined for lipid mediators or cytokines. In other experiments, infected lung cells were re-suspended in media containing monensin (0.1%, Ebioscience) in the presence or absence of 100 µg ml⁻¹ irradiated Mtb H37Rv at 10⁶ cells per well in 96-well plates and incubated at 37 °C, 5% CO₂. 5 h later cells were surface-stained, fixed/permeabilized and intracellular staining (ICS) performed.