| Title: | High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging |
| Author(s): | Park MC; Hur JY; Cho HS; Park SH; Suh KY; |
| Address: | "School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Korea" |
| ISSN/ISBN: | 1473-0189 (Electronic) 1473-0189 (Linking) |
| Abstract: | "Extracting single-cell information during cellular responses to external signals in a high-throughput manner is an essential step for quantitative single-cell analyses. Here, we have developed a simple yet robust microfluidic platform for measuring time-course single-cell response on a large scale. Our method combines a simple microwell-based cell docking process inside a patterned microfluidic channel, with programmable time-course live-cell imaging and software-aided fluorescent image processing. The budding yeast, Saccharomyces cerevisiae (S. cerevisiae), cells were individually captured in microwells by multiple sweeping processes, in which a cell-containing solution plug was actively migrating back and forth several times by a finger-pressure induced receding meniscus. To optimize cell docking efficiency while minimizing unnecessary flooding in subsequent steps, circular microwells of various channel dimensions (4-24 microm diameter, 8 microm depth) along with different densities of cell solution (1.5-6.0 x 10(9) cells per mL) were tested. It was found that the microwells of 8 microm diameter and 8 microm depth allowed for an optimal docking efficiency (>90%) without notable flooding issues. For quantitative single-cell analysis, time-course (time interval 15 minute, for 2 hours) fluorescent images of the cells stimulated by mating pheromone were captured using computerized fluorescence microscope and the captured images were processed using a commercially available image processing software. Here, real-time cellular responses of the mating MAPK pathway were monitored at various concentrations (1 nM-100 microM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics" |
| Keywords: | "Equipment Design High-Throughput Screening Assays/*instrumentation/methods Image Processing, Computer-Assisted/methods MAP Kinase Signaling System Microfluidic Analytical Techniques/*instrumentation/methods Saccharomyces cerevisiae/*cytology/enzymology Si;" |
| Notes: | "MedlinePark, Min Cheol Hur, Jae Young Cho, Hye Sung Park, Sang-Hyun Suh, Kahp Y eng Research Support, Non-U.S. Gov't England 2010/10/20 Lab Chip. 2011 Jan 7; 11(1):79-86. doi: 10.1039/c0lc00114g. Epub 2010 Oct 19" |
