# characteristics.description[].text
Protocols: Bronchoalveolar lavage (BAL) was performed on infants undergoing rigid bronchoscopy for suspected airway abnormalities at Royal Manchester Children’s Hospital, using a ventilating Storz bronchoscope under sevoflurane inhalational anesthesia. In adults, BAL was performed during flexible bronchoscopy for investigation of persistent unexplained cough or hemoptysis, as previously described (PMID: 24686157). In all participants, lavage was performed with sterile 0.9% sodium chloride solution. Exclusion criteria included any febrile illness within the last 14 days, receipt of antibiotics within previous six weeks, any evidence of immunodeficiency, immunosuppressive medication, previous close contact with an individual with TB, family history of TB, atopic disease, asthma, any chronic lung disease, any chronic inflammatory disease, or surgery involving general anaesthetic within the last four weeks. Additional exclusion criteria for infant participants included preterm delivery (less than 37 weeks gestation), low birth weight (less than 2.5kg) or congenital malformation. Peripheral blood was also obtained from all participants for interferon gamma release assay (QuantiFERON-TB Gold Plus, QIAGEN) which was performed as per manufacturer’s instructions. Alveolar macrophages isolation & culture. BAL was transported to the laboratory on ice within one hour of acquisition. Mucous was disrupted by the addition of a fourfold volume of 0.1% dithiothreitol (Sigma-Aldrich) followed by gentle rocking for 15 minutes at room temperature. The suspension was filtered serially through sterile 150um and 50um filters (CellTrics) and diluted twofold with phosphate-buffered saline (PBS). Cell pellets were obtained by centrifugation, and live Alveolar macrophages were counted by microscopy using a hemocytometer based on morphology and trypan blue exclusion. Cells were either stained immediately for flow cytometry or prepared for culture by suspension in RPMI 1640 Medium (Sigma-Aldrich) supplemented with 10% Fetal Calf Serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine, and then seeded in 24-well tissue culture plates (Corning Inc.). Following one hour of incubation at 37oC/5% CO2, non-adherent cells were removed by vigorous PBS washing, and adherent cells were cultured for downstream application. Ethical approval and participant recruitment. The research protocol was approved by the NHS Research Ethics Committee (Reference 14/SW/0100) and Manchester University NHS Foundation Trust Research Office (Reference R03601). Written informed consent was obtained from adult participants and the parents/legal guardians of participating infants. Information recorded during the recruitment of all participants included demographics and medical history. There are two experimental factors: 1. adult versus infant and 2. M. tuberculosis (Mtb) infected versus uninfected freshly isolated Alveolar macrophages (AMs). For infection of AMs following purification by adherence and PBS washes as described in the sample collection protocol, AMs were incubated with Mtb H37Rv in antibiotic-free media at the desired multiplicity of infection (MOI) of 5:1 (5 bacilli per 1 macrophage) for 24 hours at 37°/5% CO2. The MOI and incubation period chosen were determined by preliminary qPCR time course experiments that identified the expression of genes considered to be important in intracellular mycobacterial control such as STAT1, IRGM1 and NOS2 (PMID:23516984). Lysate from AM s was stored at -80oC following cell disruption with buffer RLT (QIAGEN) containing 1% β-mercaptoethanol (Sigma-Aldrich). RNA was isolated from lysates using RNeasy Micro Kit (QIAGEN) according to the manufacturer’s instructions. RNA was quantified using a Qubit 2.0 Fluorimeter (Thermo Fisher Scientific). RNA samples were assessed using a 2200 TapeStation (Agilent Technologies) and deemed of acceptable quality if they had an RNA Integrity Number (RIN) of greater than 8.0 Truseq stranded mRNA Assay. Paired end.