Figure 3. MSC-CM^IEC-6(IR) reduces apoptosis of intestinal epithelial cells after radiation in vivo and in vitro.

(A) Apoptosis was assayed by TUNEL staining 3 days after irradiation. Scale bars 50 μm. (B) Quantification of TUNEL-positive cells. n = 3 in each group. The number of positive cells in 5 crypts was scored in 100 crypts per section and reported as mean ± SD. *, P < 0.05 versus IR + DMEM-F12, #, P < 0.05 versus IR + MSC-CM^IEC-6(NOR). (C) Apoptosis of irradiated IEC-6 was assayed by TUNEL staining 3 days after radiation. Scale bars 25 μm. (D) Quantification of TUNEL-positive IEC-6 cells. Data are reported as means ± SD for 10 random fields per wells from four replicate wells per group. **, P < 0.05 versus IR + DMEM-F12, ##, P < 0.05 versus IR + MSC-CM^IEC-6(NOR). FOV, field of view. (E) Apoptosis and cell death of IEC-6 were evaluated quantitatively by flow cytometry after PI/Annexin V staining. The right lower quadrant (RLQ) contains early apoptotic cells, and the upper right quadrant (RUQ) contains late apoptotic and necrotic cells. The proportions of cells in RLQ + RUQ were as follows: Control: 14.21%; IR + DMEM-F12: 53.15%; IR + MSC-CM^IEC-6(NOR): 45.26%; IR + MSC-CM^IEC-6(IR): 22.64%. (F) The percentage of total apoptotic cells and dead cells under each condition from panel E are shown. (G) The ratio of apoptosis IEC-6 cells was determined by PI/Annexin V staining at 1, 3, 5, 7 days after radiation. Data represent means ± SD of three independent experiments. **, P < 0.05 versus IR + DMEM-F12.