Introducing specific antibodies into electropermeabilized cells is a valuable tool for eliminating specific cell functions
✍ Scribed by E. J. Verspohl; Inge Kaiserling-Buddemeier; Andrea Wienecke
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 206 KB
- Volume
- 15
- Category
- Article
- ISSN
- 0263-6484
No coin nor oath required. For personal study only.
✦ Synopsis
A technique is established for the role of intracellular proteins to be eliminated and thereby gives information about their speci®c role in signal transduction within cells. Rat pancreatic islets as well as INS-1 cells (an insulin secreting cell line) were electrically permeabilized in order to introduce high molecular weight compounds. Optimized conditions were ®ve exposures with 15-s intervals, t 200 msY an electric ®eld of 1 . 36 kV per 0 . 4 cm in a speci®c permeabilization buer at a calculated Ca concentration of 5 Â 10 À8 M. In electroporation control experiments the spectrophotometrically measured uptake of the cell membrane-impermeable propidium iodide, FITC-labelled dextran MW $ 4000 and FITC-labelled antibodies MW $ 150Y000 was established as being 81Á5 +5Á0, 82Á7 + 3Á0 and 81Á0 + 1Á0 per cent of maximum, respectively. These data were corroborated qualitatively by visualizing microscopically the ¯uorescence of the FITC-labelled compounds in islets as well as in INS-1 cells. The cells appear to reseal since control experiments indicated a short-lived out¯ow of lactate dehydrogenase (MW of 140,000 which is similar to that of antibodies) and of insulin for the ®rst 15±20 min. After electroporation the cells were functionally intact, i.e. responded to the stimulus carbachol (CCh). Only 18Á0 +10Á1 per cent of cells had not resealed after 2 h (propidium iodide uptake measured at various time intervals after electroporation). As was shown recently the eect of speci®c compounds such as CCh and CCK 8 on insulin release was eliminated selectively by antibodies against speci®c G proteins thus proving this method to be a valuable tool. In conclusion, adding antibodies to electrically permeabilized cells is a valuable tool for eliminating a speci®c cell function in order to elucidate the speci®c role of intracellular compounds. This method can probably be used for testing the speci®c role of other proteins in cell functions.