NCMECs were fixed in 10% neutral buffered formalin; then washed with PBS and treated with 0.2% Triton X-100 for 10 min; then TUNEL reaction mixtures were added and incubated for 1 h at 37°C after blocked with normal horse serum for 30 min. Cell nuclei were stained with DAPI (ZLI-9557; ZSGB-BIO, Beijing, China). Photographs were obtained using an Olympus BX63 fluorescence microscope (Olympus America Inc., Center Valley, PA, USA) at 10× magnification. The number of TUNEL-positive cells and total cells were counted in each field using ImageJ software. The percentage of TUNEL-positive cells to total cells was calculated.

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