2.2. Anchorage-Independent Soft-Agar Colony Formation Assay

To evaluate the soft-agar colony-forming ability of HME cells, Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% Fetal Bovine Serum (FBS, GE Healthcare, Chicago, IL, USA) (v/v) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (GIBCO/Thermo Fisher, Waltham, Massachusetts, United States) was used (1× DMEM). Specifically, a layer of 0.5% noble agar (Sigma Aldrich) in DMEM + 10% FBS (diluted from 2× DMEM + 20% FBS) was first laid in tissue culture plates and allowed to solidify at room temperature as the “base agar”. Subsequently, HME cells of interest were mixed with 0.25% noble agar in DMEM + 10% FBS as the “cell agar” laid on top of the “base agar” layer. Once the “cell agar” solidified, the top medium constituted of DMEM, 10% FBS, 0.5 μg/mL hydrocortisone and 5 μg/mL insulin was added on top. The plates were cultured under standard tissue incubation conditions. The media on top was refreshed every week of culturing. The colonies are visualized, usually after about 2 weeks of culturing, by normal light microscopy or by staining with methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Sigma Aldrich) per manufacturer’s protocol. The colonies were photographed using an Olympus SZX16^® Research Stereo Microscope with a 3.2× objective. For the purpose of extraction of DNA, RNA and proteins from soft agar cultures, a similar protocol was employed with the following modifications: low melting point (LMP) agarose (Invitrogen/Thermo Fisher, Waltham, MA, USA) was used instead of noble agar; the “base agar” consisted of 0.65% LMP agar in DMEM and 10% FBS, and the “cell agar” consisted of HME cells of interest in 0.325% LMP agar in the same medium as the base layer; all solidification steps were done at 4 °C for 15 min, and the same “top media” were used for culturing. Cultures were harvested after 3 days for sample preparation.

To visualize the apoptotic cells in the soft agar, a modified propidium iodide (PI) staining method was used. At the time of assay, the top medium was changed to that containing 10 µL/mL propidium iodide (PI; Sigma Aldrich) and the assay incubated in the dark at 37 °C for 30 min. The apoptotic cells, which stain positive for PI, were visualized and photographed using Olympus SZX16^® Research Stereo Microscope with a 3.2× objective. The resultant images were analyzed using ImageJ software (NIH).