Sterile 70nm cell strainers

BALF was obtained from all patients at room temperature. Briefly, a flexible bronchoscope was wedged into a sub-segmental bronchus of a predetermined region of interest based on radiographical findings. A BAL technique was performed by instilling a total of 180 ml of normal saline in 60-ml aliquots, each retrieved by low suction. Samples were filtered through sterile 70-nm cell strainers (BD Biosciences, San Jose, CA, USA) and centrifuged at 500 × g or 5 min at 4°C. Cell pellets were washed and re-suspended with cold PBS. Total cell count and cell viability were subsequently assessed using Trypan blue (ICN Pharmaceuticals, Costa Mesa, CA, USA). A total of 1–1.5 million cells were centrifuged and cell pellets were homogenised in TriReagent™ (MBL) for total RNA, or RIPA buffer (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitors; Pierce/Thermo Fisher Scientific) for SDS-PAGE/western blot analysis, followed by storage at −80°C. Differential cell population count was analysed by May-Grunewald-Giemsa staining as previously described (19 (link)).