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Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

ABSTRACT: The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as viral transfection and lipofection, are not realizable with a high degree of spatial selectivity. Here, we have used a focused femtosecond laser beam to create a small transient hole in the cellular membrane (optoporation) in order to inject nanomolar concentrations of rhodamine phalloidin (an impermeable dye molecule for staining filamentous actin) into targeted living mammalian cells (both HEK and primary cortical neurons). Following optoporation, the dye bound to the intracellular actin network and rise in fluorescence intensity was observed. Theoretical dynamics of the dye's diffusion is discussed, and numerical simulations of diffusion time constants are found to match well with experimental values.

SUBMITTER: Dhakal K

PROVIDER: S-EPMC4197434 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

Dhakal Kamal K Black Bryan B Mohanty Samarendra S

Scientific reports 20141015

The selective insertion of foreign materials, such as fluorescent markers or plasmids, into living cells has been a challenging problem in cell biology due to the cell membrane's selective permeability. However, it is often necessary that researchers insert such materials into cells for various dynamical and/or drug delivery studies. This problem becomes even more challenging if the study is to be limited to specific cells within a larger population, since other transfection methods, such as vir ...[more]

PMID: 25315642

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Project description:High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.

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Dataset Information

Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

Publications

Introduction of impermeable actin-staining molecules to mammalian cells by optoporation.

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