High-throughput flow cytometry analysis in yeast and human cells

Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols.
Conclusion:
Our protocols allow fast, high-throughput analyses of key replication parameters for both budding yeast and human cells in a 96-well plate format. We also present the first successful detection of yeast nuclear proteins by flow cytometry. A proof-of-principle application of antibody-based FLAG-tag detection allows qualitative and quantitative comparison of the association of various proteins to chromatin. Utilising our optimised protocols, we not only show that the central protein complexes of origin licensing, ORC and MCM2-7, are recruited in the same manner to yeast and human chromatin. We also provide measures to reduce hands-on time, reagent consumption, overall cost per well, and plastic waste. In addition, signal to noise ratios, throughput, and uniformity of protocols were optimised. Taken together, we provide a comprehensive tool kit for efficient and fast cytometric analysis of DNA replication parameters in cellula.
Notes:
Our protocols try to minimize the ecological footprint of flow cytometry experiments by reducing hands-on time, reagent consumption, overall cost per well, and plastic waste. Please get in contact for template files and analysis files, as well as all files that cannot be downloaded. The database failed to upload some data properly. Controls included: unstained samples, single stained samples, various antibodies coupled to different dyes, mutant strains, cell cycle arrests and biological replicates