Bead Loading Proteins and Nucleic Acids into Adherent Human Cells
| Autor: | Timothy J. Stasevich, Amanda Koch, Matthew N. Saxton, Gabriel Galindo, Charlotte A. Cialek |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
General Immunology and Microbiology
Macromolecular Substances General Chemical Engineering General Neuroscience Cell RNA DNA Article General Biochemistry Genetics and Molecular Biology Bead (woodworking) Cell membrane chemistry.chemical_compound Membrane medicine.anatomical_structure chemistry Nucleic Acids medicine Nucleic acid Biophysics Animals Humans Peptides Function (biology) Plasmids |
| Zdroj: | J Vis Exp |
| ISSN: | 1940-087X |
| DOI: | 10.3791/62559-v |
| Popis: | Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing proteins into a cell across its membrane is difficult. The limited selection of current methods struggles with low efficiency, requires expensive and technically demanding equipment, or functions within narrow parameters. Here, we describe a relatively simple and cost-effective technique for loading DNA, RNA, and proteins into live human cells. Bead loading induces a temporary mechanical disruption to the cell membrane, allowing macromolecules to enter adherent, live mammalian cells. At less than 0.01 USD per experiment, bead loading is the least expensive cell loading method available. Moreover, bead loading does not substantially stress cells or impact their viability or proliferation. This manuscript describes the steps of the bead loading procedure, adaptations, variations, and technical limitations. This methodology is especially suited for live-cell imaging but provides a practical solution for other applications requiring the introduction of proteins, beads, RNA, or plasmids into living, adherent mammalian cells. |
| Databáze: | OpenAIRE |
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