期刊
LAB ON A CHIP
卷 10, 期 18, 页码 2449-2457出版社
ROYAL SOC CHEMISTRY
DOI: 10.1039/c004857g
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资金
- NSERC
- FQRNT
- CIHR
- Genome Canada
- Genome Quebec
- Canadian Institutes of Health Research [MSH 69117]
- Canada Research Chair
- Iranian Ministry of Health and Education
- Spanish Ministry of Education and Science
High resolution live cell microscopy is increasingly used to detect cellular dynamics in response to drugs and chemicals, but it depends on complex and expensive liquid handling devices that have limited its wider adoption. Here, we present a microfluidic perfusion system that is built without using specialized microfabrication infrastructure, simple to use because only a pipette is needed for liquid handling, and yet allows for rapid media exchange and simultaneous fluorescence microscopy imaging. Yeast cells may be introduced from a culture, or spotted as arrays on a coverslip, and are sandwiched with a 20 mu m thick track-etched membrane. A second coverslip and a mesh with 120 mu m porosity are placed on top, forming a microfluidic conduit for lateral flow of solutions by capillary effects. Solutions introduced through the inlet flow through the mesh and chemicals diffuse vertically across the membrane to the cells trapped below. Solutions are exchanged by adding a new sample to the inlet. Using this system, we studied the dynamic response of F-actin in living yeast expressing Sac6-EGFP-a protein associated with discrete F-actin structures called patches''-to the drug latrunculin A, a well known inhibitor of actin polymerization. We observed that the patches disappeared in 85% of the cells within 5 min, and re-assembled in 45 min following exchange of the drug with media. The perfusion system presented here is a simple, inexpensive device suited for analysis of drug dose-response and regeneration of single cells and arrays of cells.
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