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 x 250 microm(2) with a height of 60 microm. 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.
Day
Daniel
D
Centre for Micro-Photonics, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Vic, Australia. dday@swin.edu.au
Pham
Kim
K
Ludford-Menting
Mandy J
MJ
Oliaro
Jane
J
Izon
David
D
Russell
Sarah M
SM
Gu
Min
M
D000818:Animals; D017951:Antigen Presentation; D002450:Cell Communication; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D017154:Stromal Cells / Q000166:cytology; D013950:Thymus Gland / Q000166:cytology; D013997:Time Factors