75 mm polystyrene round bottom test tubes

Cells were cultured in a 35 cm culture dish until 75% confluence, and then trypsinized and resuspended in phosphate-buffered saline (PBS) with 1.5% FBS, 25 mM HEPES pH 7.0, and 1 mM EDTA (Sigma-Aldrich, USA). Cells were then transferred into 75 mm polystyrene round-bottom test tubes (BD Falcon, USA) at a concentration of 1 × 10⁶ cells/ml, stained with fluorescence-conjugated antibodies (anti-CD13-APC, anti-CD24-APC-eFlour 780, anti-CD44-eFluor 450, anti-CD133-PE, anti-EpCAM-FITC, and 7-AAD) as previously described [12 (link)]. Cells stained with isotypematched antibodies (BD Biosciences, USA) served as negative controls. Flow cytometer analysis was performed on a BD FACSCanto-II Digital Flow Cytometry Analyzer (USA) using FlowJo (Tree Star, USA) software.

The NSCLC cell lines (A549 and H2170) were harvested upon incubation with 0.25% trypsin (Life Technologies, Foster City, CA, USA) and washed with phosphate-buffered solution with 2% FBS. The CD166-PE and EpCAM-FITC (BD Biosciences, San Jose, CA, USA) antibodies were used for CSC identification by flow cytometry. Briefly, cells were trypsinised, counted by a haemocytometer and transferred to 75-mm polystyrene round-bottom test tubes (BD Falcon, NJ, USA) at a cell concentration of 1×10⁶ cells/ml and subsequently stained with 10 µl of antibodies in the dark at 4°C. The cells were then washed and filtered through a 40-µm cell strainer to obtain a single-cell suspension before sorting. The expression of cancer stem cell markers (CD166 and EpCAM) was analysed and sorted using FACSAria III (BD, Biosciences). Gating used for the sorting of CD166⁺/EpCAM⁺ (Q2) and CD166⁻/EpCAM⁻ (Q3) NSCLC cell lines is depicted in Fig. 3.