Fig. 8
Effect of bafilomycin A1 on the development and ischemic tolerance of ACMs. 1 mL of CMDF cell suspension was divided into four groups (250 µL each). The CMDF cells were pre-exposed to normoxia (Norm) or ischemia (Isch) in the presence or absence of 100 nM bafilomycin A1 (Baf) for 90 min at 37 °C, washed out with centrifugation to remove Baf and subsequently cultured in the absence of Baf. The cells without Baf treatment were given the same volume of DMSO. a Left panel shows the cell number of beating ACMs cultured for 5 days. Data are mean ± SEM (n = 3 cell preparations). **p < 0.001. Right panels show the phase contrast microscopy of beating ACMs (bars 50 µm). b Phase contrast images of CMDF cells with or without pre-exposure to ischemia in the presence of Baf and cultured in the continued presence of Baf for 4 days (bar 50 µm). c Left panels show the confocal laser scanning microscopy of double-immunostaining of LC3 (green) and α-actinin (red), DAPI staining (blue) and DIC images of ACMs cultured in the absence of Baf for 5 days. The conditions of pre-treatment of CMDF cells are the same as (a) (bar 50 µm). Right panel shows the immunofluorescent signals for the estimated membrane-bound LC3. Data are mean ± SEM. (n = 8 cells obtained from 3 mice). **p < 0.001, *p < 0.005