Fig. 2

The measurement of mitotic and apoptotic p75⁺ ENSC fractions in NSM and SRM. A Dispersed enterocytes were first labeled with CFSE and then grown in NSM and SRM, respectively, for 24 h. Cells were subjected to surface-stained with anti-p75 and Alexa Fluor 647-conjugated secondary antibody, and analyzed by flow cytometry. Defined by isotype controls, the population of p75⁺ ENSCs was gated (green) in contour plots (upper panels), and presented in histogram plots (lower panels). On day 0, p75⁺ ENSCs exhibited a single peak of CFSF fluorescence intensity. After 24-h cultivation in NSM or SRM, a fraction of p75⁺ ENSCs underwent mitosis and led to the second peaks (G2) in histograms, which had the reduction of CFSE mean fluorescence intensity (MFI) by half as compared to the MFI of the original peaks (G1). Representative datasets shown were derived from the same batch of dispersed enterocytes, illustrating the calculation process of mitotic p75⁺ ENSC fractions in NSM and SRM, respectively. B Within 24-h cultivation, the mitotic fractions of p75⁺ ENSCs did not differ between NSM and SRM culture conditions (p = 0.515). C Cells nurtured in NSM or SRM were subjected to apoptosis assays. The system was setup using neurosphere cells stained after cell dissociation by Accutase (left panels). The population of p75⁺ ENSCs was gated (green) and analyzed in histogram plots (right panels). The figures presented were representative histograms of day 6 NSM and SRM-cultivated cells with annexin V staining after (n = 3) or before cell detachment (n = 6), showing significantly higher fractions of apoptotic p75⁺ ENSCs harvested from NSM (p = 0.010) and SRM (p = 0.003) cultures in the case of annexin V staining after cell detachment. The fractions shown were mean ± standard error of mean with their ranges in parentheses. D Neurosphere cells were stained with annexin V before cell detachment on days 3 (D3) and 6 (D6), respectively, SRM led to a higher fraction of apoptotic p75⁺ ENSCs than NSM at both time points. Apoptosis of p75⁺ ENSCs did not differ between D3 and D6 in NSM (p = 0.263) and SRM (p = 0.248)