Dataset Information

Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

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.

SUBMITTER: Godino E

PROVIDER: S-EPMC10367129 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

Godino Elisa E Restrepo Sierra Ana Maria AM Danelon Christophe C

ACS synthetic biology 20230508 7

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 fl ...[more]

PMID: 37155828

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Project description:Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.

Dataset Information

Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

Publications

Imaging Flow Cytometry for High-Throughput Phenotyping of Synthetic Cells.

Similar Datasets

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