Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies
✍ Scribed by Gary R. Bright; Ning-Tsu Kuo; David Chow; Susan Burden; Catherine Dowe; Ronald J. Przybylski
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 827 KB
- Volume
- 24
- Category
- Article
- ISSN
- 0196-4763
No coin nor oath required. For personal study only.
✦ Synopsis
A method is described to introduce by electroporation membrane-itnpermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrierbased dyes (e.g., fluorescein-labeled dextran), large macromolecules (e.g., antibodies), and metabolic precursors (e.g., 32P-ATP) with high efficiency. Conditions are relatively independent of cell type. Electroporation with three pulses of 300 volts at 540 pF capacitance at 4°C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 f 0.15 mm. Suitable poration medium includes calcium-magnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered su-crose. Potential use in fluorescence imaging and metabolic studies is shown with DNA synthesis, cell replication, cell substratum attachment, 32P-ATP phosphorylation, and insulin-mediated increases in glucose uptake and its suppression by antiphosphotyrosine and antiglucose transporter protein antibodies. The ability to load foreign molecules into large numbers of adherent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic imaging, as well as with conventional biochemical and physiological techniques. o 1996 mey