High-throughput screening and sorting of optogenetic cell samples
Kathrin Brenker1, Barbara Di Ventura2, Giada Forlani2,3, Luis Köbele1, Diana Ordonez4, Malte Paulsen6 and Daniel Schraivogel4
1opto biolabs GmbH, Freiburg, No State, Germany, 2Signalling Research Centers BIOSS and CIBSS, and Institute of Biology II, Faculty of Biology, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany, 3Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-Universität Freiburg, Freiburg, Germany, 4European Molecular Biology Laboratory, Heidelberg, Germany, Heidelberg, Germany, 5NovoNordisk Foundation Center for Stem Cell Research – DanStem, Kopenhagen, Denmark
Flow cytometry is a very powerful analysis technique, rapidly analyzing and sorting thousands of cells at single-cell resolution. Recently, image–enabled cell sorting (ICS) was established. ICS allows the rapid identification and isolation of cells based on conventional fluorescence parameters and image-derived parameters. Thanks to that, cells with specific morphological traits can be isolated from heterogeneous populations and used for downstream analysis or further culturing.
The nucleus is a complex and dynamic eukaryotic organelle. To guard and organize the genetic information, proteins enter and exit the nucleus in a highly organized manner. The optogenetic tools LINuS and LEXY allow controlling the nuclear import and export of proteins of interest with blue light, respectively. They have been applied to a variety of eukaryotic proteins and their response time (second-minute-range) is perfectly suited for cytometric analyses.
Here, we equipped an ICS-instrument with specialized illumination devices to enable optogenetic-ICS (optoICS) for the first time. We were able to induce import and export in LINuS- and LEXY-expressing cells, respectively and to quantify the degree nuclear translocation over time using optoICS. This powerful technique enabled us to sort thousands of cells per second based on their degree of translocation as well as traditional cytometric parameters, like total fluorescence.
In summary, we present a novel technique for optogenetics that enables the stimulation, analysis, imaging and sorting of cell samples activated by light. optoICS can not only be used to analyze nuclear translocation, but also other light-induced processes such as cell division, membrane localization and many more.