Cellular characterisation of the basal layer of 2D enteroids. mRNA expression of A α-smooth muscle actin and B desmin in freshly isolated villi and in 2D enteroids grown on uncoated and Matrigel coated cell culture plastic at day 5 of culture. Data is represented as the mean of three independent experiments and 95% CI. Immunolocalisation of basal mesenchymal cell types in 2D enteroids grown on Matrigel coated cell culture plastic (C–F) or transwells (G–I). After removal of the apical cells, the cells are stained for C, D vimentin (mesenchyme, green). E, F To demonstrate the subepithelial layer, the cells are permeabilised with Triton X100 on day 2 and double stained for E-cadherin (epithelial cells with adherens junctions, green) and vimentin (mesenchyme, red). Vimentin expression is evident in the sub-epithelial layer. Images from 3 independent experiments. All cells are counterstained with DAPI (nuclei, blue). Scale bars: 50 µm. (G-J) Immunolocalisation of F-actin (red) and DAPI (nuclei, blue) in 2D enteroids grown on Matrigel coated transwells at day 4 of culture. Z- stack of G Apical, H Middle and I, J Basal slices. I, J DAPI stained nuclei in the basal slices demonstrate the presence of nuclei in the subepithelial layer (white arrowheads), whereas long F-actin filaments from mesenchymal cells (I) cross the basal layer slice (white arrows). The lines on the main photo indicate the image area represented in the transverse slices at the bottom and side of each photo. Representative images of 1 experiment. Scale bars: 15 µm.