Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Cytochemical localization of dipeptidyl peptidase II (DPP-II) in mature guinea pig sperm.

Guinea pig sperm contain the exopeptidase dipeptidyl peptidase II (DPP-II). Our purpose was to localize this enzyme in sperm at the ultrastructural level using lysylalanyl-4-methoxy-beta-naphthylamide which is the specific synthetic substrate for DPP-II. In substrate incubated sperm, reaction product was located within the acrosome. It was concentrated over the light staining area which forms the prominent dorsal bulge of the acrosome. Within this area there are spherical zones of moderate electron density which had little or no reaction product. Control sperm did not have reaction product. We conclude that DPP-II is restricted to a compartment within the guinea pig sperm acrosome. </jats:p