Flow cytometry to identify leukocyte sub-populations in blood and induced sputum in asthmatic and healthy volunteers exposed to diesel exhaust

Methods Mild asthmatics and normal controls were recruited as study subjects. This crossover study was double-blinded, randomized and counter-balanced to the order of three conditions: diesel exhaust with anti-oxidant, diesel exhaust with placebo, or filtered air with placebo. The subjects were exposed to either filtered air or diesel exhaust (300 ug PM2.5/m ) in a state-of-the-art diesel exhaust exposure facility. An anti-oxidant, N-acetylcysteine (600 mg), or a placebo was taken orally for five days preceding, and on the day of the exposure. Each subject was exposed to each of the three conditions. Peripheral blood samples were taken pre-exposure, and also at 2, 6, and 30 hours after the beginning of exposure. Sputum induction was performed by inhalation of hypertonic saline according to ATS guidelines pre-exposure, and also at 6, and 30 hours after the beginning of exposure. FACSCanto II (BD Biosciences) was used for flow cytometry. A 5-colour, 12-marker (CD3/CD9/ CD14/CD16/CD19/CD20/CD45/CD56/CD83/CD206/ CD326/HLA-DR) combination was used to identify dendritic cells, macrophages, monocytes, neutrophils, eosinophils, and bronchial epithelial cells. Direct immunolabelling was performed on whole peripheral blood. After incubation, red blood cells were lysed. Remaining cells were washed and resuspended in PBS with 0.5% paraformaldehyde. Sputum plugs were homogenized with 0.1% DTT, filtered, and then centrifuged to remove supernatant. Sputum cells were resuspended in PBS at 1 million per mL. Direct immunolabelling was performed. After incubation, cells were washed and resuspended in PBS with 0.5% paraformaldehyde. Spectral compensation for flow cytometry was performed using an automatic calibration technique (BD CompBeads). Cellular debris was eliminated on the SSC/FSC scattergram. A gating strategy was designed to identify the leukocyte sub-populations and bronchial epithelial cells. Surface markers were chosen based on differential cell-specific expression according to existing literature.