High-throughput single-cell quantification using simple microwell-based cell docking and programmable time-course live-cell imaging.
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 µm diameter, 8 µm depth) along with different densities of cell solution (1.5-6.0 × 10(9) cells per mL) were tested. It was found that the microwells of 8 µm diameter and 8 µm 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 µM) of mating pheromone at single-cell resolution, revealing that individual cells in the population showed non-uniform signaling response kinetics.
Park
Min Cheol
MC
School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-742, Korea.
Hur
Jae Young
JY
Cho
Hye Sung
HS
Park
Sang-Hyun
SH
Suh
Kahp Y
KY
D004867:Equipment Design; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods; D020935:MAP Kinase Signaling System; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods