Print Email Facebook Twitter Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells Title Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells Author Godino, E. (TU Delft BN/Christophe Danelon Lab; Kavli institute of nanoscience Delft) Restrepo Sierra, A.M. (TU Delft BN/Gijsje Koenderink Lab; Kavli institute of nanoscience Delft) Danelon, C.J.A. (TU Delft BN/Bionanoscience; INSA Toulouse; Kavli institute of nanoscience Delft) Department BN/Bionanoscience Date 2023 Abstract The reconstitution of basic cellular functions in micrometer-sized liposomes has led to a surge of interest in the construction of synthetic cells. Microscopy and flow cytometry are powerful tools for characterizing biological processes in liposomes with fluorescence readouts. However, applying each method separately leads to a compromise between information-rich imaging by microscopy and statistical population analysis by flow cytometry. To address this shortcoming, we here introduce imaging flow cytometry (IFC) for high-throughput, microscopy-based screening of gene-expressing liposomes in laminar flow. We developed a comprehensive pipeline and analysis toolset based on a commercial IFC instrument and software. About 60 thousands of liposome events were collected per run starting from one microliter of the stock liposome solution. Robust population statistics from individual liposome images was performed based on fluorescence and morphological parameters. This allowed us to quantify complex phenotypes covering a wide range of liposomal states that are relevant for building a synthetic cell. The general applicability, current workflow limitations, and future prospects of IFC in synthetic cell research are finally discussed. Subject cell-free expressiondirected evolutionin vitro transcription−translationliposomeminimal cellsynthetic cell To reference this document use: http://resolver.tudelft.nl/uuid:77636615-189a-40f4-b2bf-ca9da8c24b2d DOI https://doi.org/10.1021/acssynbio.3c00074 ISSN 2161-5063 Source ACS Synthetic Biology, 12 (7), 2015-2028 Part of collection Institutional Repository Document type journal article Rights © 2023 E. Godino, A.M. Restrepo Sierra, C.J.A. Danelon Files PDF acssynbio.3c00074.pdf 10.19 MB Close viewer /islandora/object/uuid:77636615-189a-40f4-b2bf-ca9da8c24b2d/datastream/OBJ/view