Fig 2. WEHI-YH2 feeder layer co-culture stimulates stem cells in colon crypt cultures.

Epithelial cells were purified using EpCAM-PE/Cy7, CD44-PE and CD24-APC antibodies. (A) The CD44⁺CD24⁺ cells in quadrant 2 in the bivariate plot were gated. A further gate on SSC^loCD44^hi subset followed by CD24^{sub-lo/lo/med} subsets were used to purify colon epithelial cells into 3 further subsets: EpCAM⁺SSC^loCD44^hiCD24^sub-lo, EpCAM⁺SSC^loCD44^hiCD24^lo and EpCAM⁺SSC^loCD44^hiCD24^med. (B) Representative images of colonoids formed after the 3 subsets of colon cells were embedded in Matrigel and co-cultured with a monolayer of WEHI-YH2 cells for 8 days. Scale bar = 50 μm. (C) The formation efficiency (%) of colonospheres and colonoids in day 8 culture. Error bars: SEM n≥3, 1-way ANOVA, *p<0.05.