6 cm ultra low attachment dishes

Cells were plated in 6 cm ultra-low attachment dishes (Corning, Lowell, MA, USA); on the seventh day, the mammospheres were counted and measured under a low power field inverted microscope. The mammosphere formation efficiency was calculated as the percentage ratio of obtained spheres and plated cells 17 (link), and was calculated by using the mean ± SD and t-test.

miPS-LLCcm cells (4 × 10⁴) were seeded on 6-cm ultra-low attachment dishes (Corning Inc., Corning, NY, USA) or on 24 well ultra-low attachment dishes (Corning Inc., Corning, NY, USA) (1 × 10⁴ cells/well) in FBS-free DMEM supplemented with Insulin-Transferrin-Selenium-X (ITS-X; 1/100 v/v) (Life Technologies, Carlsbad, CA, USA), 0.1 mM NEAA, 2 mM L-glutamine 50 U/mL penicillin/streptomycin (Wako, Osaka, Japan) and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). After 5–7 days, the number of spheres was counted, and images were acquired using CKX41 inverted microscope (Olympus, Tokyo, Japan) or an IX81 inverted microscope (Olympus, Tokyo, Japan) equipped with a light fluorescence device (Olympus, Tokyo, Japan). For limiting dilution analysis, we seeded 100, 10 and 1 cells in 96-well low attachment plates (EZ-BindShut^TMSP, Asahi Glass Co., Ltd., Tokyo, Japan) in the above medium and after 1 week of incubation stem cell frequency was calculated with software available at http://bioinf.wehi.edu.au/software/elda/index.html [58 (link)].

The cells (5 × 10⁴) were seeded on 6-cm ultra-low attachment dishes (Corning Incorporated, NY) in FBS-free DMEM supplemented with Insulin-Transferrin-Selenium-X (ITS-X) (Life Technologies, CA), 0.1 mM NEAA, 2 mM L-glutamine, and P/S. After 3–4 days, sphere-forming cells were collected by centrifuging the cells at 300 rpm for 5 minutes and resuspending them in 1× phosphate buffered saline (PBS). After aspirating the PBS, the spheres were dissociated using Accutase® cell detachment solution (Sigma, USA). Then, the cells were seeded at a density of 1 × 10⁴ cells/ml in ultra-low attachment dishes the same medium. After 4 days, spheres with a diameter of approximately 100 μm were seeded in 6-cm adherent dishes coated with 0.1% gelatin (Sigma) containing a 1:1 ratio of DMEM supplemented with 5% KSR and P/S, and CM from the primary CSCs. The medium was changed every other day, and differentiated cells were maintained for no longer than 2–3 passages before being dissociated. Differentiated cells were separated using feeder removal microbeads, an LS column, and a Midi MACS separator (Miltenyi Biotec, Singapore). The cells were subsequently maintained in the same medium supplemented with KSR for up to 5 passages.