Figure 2

Maintenance of passaged MACS+ cells on STO cells and rhythmic pacemaker activity of cultured ICC (MACS+ and passaged MACS+ cells). 60 k sorted cells were cultured for 7 days unless otherwise noted. (a) Immunofluorescence of passaged MACS+ (P-MACS+) cells with ICC markers, Kit (red) and Ano1 (green) and with co-localization (yellow). MACS+ cells were cultured on STO cells for 7 days and were passaged and sorted with MACS (P-MACS+). P-MACS+ cells were subsequently cultured on STO cells. Scale bar, 200 µm. (b,c) Growth comparison of GFP + MACS+ and P-MACS+ cells with mRNA expression of Kit (b) and DNA expression of gfp (c). (b,c: day 1, 4, 7 n = 4; day 14 n = 2). (d–f) Oscillations in intracellular Ca²⁺ concentration demonstrated the rhythmic pacemaker activity in MACS+ and P-MACS+ ICC cultures and their frequency were measured. (d) Ca²⁺ oscillation frequency of MACS+ cells cultured on STO cells or Ge at day 7 (n ≥ 5). (e) Ca²⁺ oscillation frequency of 60 k MACS+ and 60 k P-MACS+ cells cultured on STO cells at day 7, and the frequency of 15 k MACS+ cells cultured on STO cells at day 14 (n ≥ 5). (f) Representative time-course change in fluorescence intensity due to Ca²⁺ oscillation of ICC in the culture of MACS+ cells on STO cells at day 7 (Supplementary Video 1). *Samples were normalized to de-epithelialized intestine. Error bars, s.d. ***P < 0.0001, *P < 0.05.