The concentration of the cytokines, IL-1β, IL-6, and IL-8, was analyzed in sputum supernatants using BD™ CBA Flex Set System (BD Bioscience-PharMingen, San Diego, CA, USA) according to the manufacturer's instructions [29 (link), 31 (link)]. DTT (0.025%) was added to the standard curve and enzyme immunoassay buffer [32 (link)]. The lower detection limits of the cytokines were 2.3, 1.2, and 1.6 pg/mL for IL-1β, IL-8, and IL-6, respectively.
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