2.3. Sputum Induction and Processing

Sputum induction was conducted by the inhalation of a nebulised, sterile mixture of 4.5% sodium chloride (hypertonic) and salbutamol 200 µg, followed by the coughing and expectoration of airway secretions. For nebulisation, an ultrasound nebuliser (Model CUN60 Citizen System Japan Co. Ltd., Tokyo, Japan) was was used as recommended [24] with a mouthpiece that fitted an output of ∼1 mL·min⁻¹ to achieve successful sampling. The induced sputum samples collected from the respondents were kept in an icebox and further processed within two hours by using flow cytometry. The method of processing sputum samples has been previously described [25]. In short, the sputum sample was diluted with freshly prepared phosphate buffer saline and gently vortexed at room temperature for homogenisation. These steps were repeated thrice. Subsequently, the sputum samples were centrifuged at 800× g for 10 min. Next, cytospin slides of sputum cells were stained with May–Grunwald–Giemsa for the cell differentiation count. Samples with >80% of squamous epithelial cells were excluded for the flow cytometry analysis.