Protocol for in situ visualization of mitochondrial ROS and apoptosis in spatially confined cells and sample preparation for biochemical analysis

Locomotion through spatially confining spaces is an important in vivo migration mode. Here, we present a protocol for in situ visualization of mitochondrial reac-tive oxygen species and apoptosis in cancer cells during confined migration. We then detail sample preparation of confined cells for transcriptome and imm-unoblotting analysis by using transwell chambers. This approach allows in situ evaluation of a variety of cellular functions during confined migration and prep-aration of the samples of confined cells for further biochemical analysis. For complete details on the use and execution of this protocol, please refer to Cai et al.1