High-throughput microfluidics to control and measure signaling dynamics in single yeast cells.
Anders S HansenNan HaoErin K O'SheaPublished in: Nature protocols (2015)
Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. By using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, while collecting data for thousands of single cells. Compared with other protocols, the present protocol is relatively easy to adopt and of higher throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.
Keyphrases
- high throughput
- single cell
- induced apoptosis
- gene expression
- randomized controlled trial
- single molecule
- cell cycle arrest
- living cells
- transcription factor
- high resolution
- rna seq
- endoplasmic reticulum stress
- dna methylation
- high speed
- machine learning
- oxidative stress
- optical coherence tomography
- signaling pathway
- fluorescent probe
- genome wide
- cell proliferation
- big data
- stem cells
- mesenchymal stem cells
- label free
- saccharomyces cerevisiae
- bone marrow
- bioinformatics analysis