A method for prolonged imaging of motile lymphocytes

With new imaging technologies and fluorescent probes, live imaging of cells in vitro has revolutionized many aspects of cell biology. A key goal now is to develop systems to optimize in vitro imaging, which do not compromise the physiological relevance of the study. We have developed a methodology that contains non‐adherent cells within the field of view. ‘Cell paddocks’ are created by generating an array of microgrids using polydimethylsiloxane. Each microgrid is up to 250 × 250 μm2 with a height of 60 μm. Overlayed cells settle into the grids and the walls restrict their lateral movement, but a contiguous supply of medium between neighboring microgrids facilitates the exchange of cytokines and growth factors. This allows culture over at least 6 days with no impact upon viability and proliferation. Adaptations of the microgrids have enabled imaging and tracking of lymphocyte division through multiple generations of long‐term interactions between T lymphocytes and dendritic cells, and of thymocyte–stromal cell interactions.