Colony-forming efficiency (CFE) assay

Cultured cells were detached, centrifuged, single-cell suspended, and plated at a density of 700 or 1000 viable cells per well in 6-well plates containing growth-arrested 3T3 mouse fibroblasts. Similarly, to determine the clonal growth ability of GCs one day after passage, the unattached GC-enriched subpopulations were harvested and plated at a density of 1000 or 2000 viable cells per well. All experiments were performed in triplicate for each donor and condition (i.e., IL-13- and IL-13+). On day 12 or 13 after the cultures were started, the colonies were washed with phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific), fixed with ice-cold methanol for 30 min at -20°C, rehydrated in PBS, and stained for 5 min at 37°C with 2% rhodamine B (Merck KGaA, Darmstadt, Germany). After the rhodamine B had been removed, the colonies were washed with tap water until optimal staining intensity was achieved. The plates were photographed, and the total number of colonies was counted using NIS Elements software (Laboratory Imaging, Prague, Czech Republic). The total CFE (%) was calculated as follows: (total number of colonies formed at the end of growth period/total number of viable cells seeded) × 100.