Non adherent 24 well plates

The process is referred to the previous study [18 (link)]. Briefly, NSCLC cells were maintained in DMEM-F12 medium containing B27 (Sigma, 20 ng/ml) and EGF (BD Biosciences, San Jose, CA, 10 ng/ml) in non-adherent 24-well plates (Corning, NY) at 1000 cells/well for 10 days. After then, spheroids with more than 50 μm were photographed and counted. For analysis on spheroids, spheroids were collected, trypsinized and re-seeded in plates. The spheroid-derived cells were incubated until the end of each experiment, and fresh spheroids were collected for each experiments.

Cells collected by the cell sorter were cultured in non-adherent 24 well plates (Corning, New York, NY, USA) (LepR/Tom⁺ BM cells: 4781‒9849 cells/well, LepR/Tom⁻ BM cells: 18,642‒201,149 cells/well, LepR/Tom⁺ PDL cells: 83‒348 cells/well, LepR/Tom⁻ PDL cells: 9125‒61,003 cells/well) with spheroid-forming media^{22 (link),27 (link)–29 (link)} (1:2 ratio of DMEM/F-12 (1:1) (21331-020) and Human Endothelial Medium (11111-044) supplemented with 3.75% Chicken Extract, 0.1 mM β-ME, 1% Non-essential amino acids, 1% Antibiotic–Antimycotic, 1% N2, 2% B27, 20 ng/mL mouse PDGF-AA (all from Thermo Fisher Scientific), 20 ng/mL human bFGF (ReproCELL Inc., Kanagawa, Japan), 20 ng/mL mouse oncostatin M, 20 ng/mL mouse IGF-1 (all from R&D SYSTEMS), 20 ng/mL mouse EGF (Sigma-Aldrich, St. Louis, MO, USA)). After culturing for 14 days, spheroid-forming efficiency was determined. Fluorescence and phase-contrast images of spheroids were acquired using an All-in-One Fluorescence Microscope (BZ-X700) equipped with a BZ-X-Viewer, BZ-X Analyzer (all from KEYENCE, Osaka, Japan), a CFI Plan Fluor DL (4×/0.13), and a CFI Plan Fluor DL (10×/0.45) (both from Nikon, Tokyo, Japan).

Mouse sorted cells at 1 × 10³ were transferred to non-adherent 24 well plates (Corning) with spheroid-forming media^{5 (link), 7 (link), 47 (link)} [1:2 ratio of DMEM F12 (Gibco) and Human Endothelial Medium (Gibco) supplemented with 3.75% Chicken Extract (US Biological), 0.1 mM β-ME (Invitrogen), 1% Non-essential amino acids (Gibco), 1% Pen-strep (Gibco), 1% N2 (Gibco), 2% B27 (Gibco), 20 ng/mL human bFGF (R&D Systems), 20 ng/mL mouse PDGF (Peprotech), 20 ng/mL mouse oncostatin M (R&D Systems), 20 ng/mL mouse IGF-1 (Peprotech), 20 ng/mL mouse EGF (Peprotech)]. After 7 days, the spheroid efficiency was determined.