Quantitative analysis of surface area of early and late endosome.

Z-stack images were acquired using an Andor spinning disc confocal microscope with an Ixon3 electron-multiplying charge-coupled-device (EMCCD) detection system. Images were then used to perform three-dimensional surface reconstruction using Imaris x64 7.6.3 (Bitplane AG). The surpass module was used to construct the surface, followed by manual threshold adjustment. All parameters were kept constant for independent studies. Surface area was calculated from three independent cells, and the mean value was calculated to obtain the overall surface area of early endosome and late endosome.