M tuberculosis erdman

Peripheral blood mononuclear cells were stained for CD11b, CD33, CD14, HLA-DR; CD11b⁺CD33⁺CD14⁺HLA DR^-/lo MDSC and CD11b⁺CD33⁺CD14⁺HLA DR^+/hi monocytes were isolated using Beckman Coulter MoFlo flow cytometer; sorted cells were >90% positive (Supplemental Figure 1). Isolated cells (50, 000 – 80, 000) were cultured in RPMI 1640 (Gibco) and 10% human serum (MP Biomedicals) at 37⁰C and 5% CO₂ in the presence or absence of, M tuberculosis Erdman (Erdman) or M tuberculosis H37Rv whole cellular lysate (WCL) (10 µg/ml) (BEI resources) for 24 hrs. Supernatants and cells in Trizol were stored at -80°C for cytokine measurement and RNA purification, respectively. For some experiments, cells were cultured in the absence or presence of recombinant IL-27 (rIL-27) (10 ng/ml; R&D Systems).

Female C57BL/6 mice were infected intranasally with M. tuberculosis Erdman (BEI Resources, Manassas, VA, USA) 6 weeks after immunization and kept in isolators under BSL-3 containment. Frozen aliquots as received from BEI Resources were thawed at room temperature, and diluted in saline to a concentration of 1.4x10⁴ CFU/ml. Mice were anaesthetized by an intraperitoneal injection of a combination of Ketamine (50 mg/kg; Ketalar, Pfizer Itd, Kent, UK) and Xylazine (10 mg/kg; Rompun; Berkshire, UK) in saline. Each animal then received 50 μl of the inoculum, estimated to contain 700 CFU. The number of bacteria in the inoculum was confirmed by plating aliquots on 7H11 agar plates containing 10 % OADC and 0.5 % glycerol.
Four weeks after infection, animals were killed by cervical dislocation. Lungs and spleens were removed aseptically and homogenized by mechanical disruption in sterile PBS. A series of 10-fold dilutions of tissue homogenates in PBS with 0.05 % Tween 80 were plated onto 7H11 agar plates with 10 % OADC supplement and 0.5 % glycerol. Plates were incubated at 37 °C and colonies counted after 3 weeks.