A method for reversible permeabilization of isolated rat ventricular myocytes.
A method is described that enables the cell membrane of isolated rat ventricular myocytes to be permeabilized and resealed while maintaining cell viability. Streptolysin O, a cholesterol-binding cytolysin, was used to form pores in the surface membrane; subsequent incubation with 5% fetal bovine serum was used to reverse this permeabilization. The efficacy of membrane permeabilization and resealing was ascertained using a simultaneous double-staining technique using propidium iodide, a marker for cells with permeabilized membranes, and fluorescein diacetate, a marker for viable cells. This procedure allowed a distinction to be made between dead cells, unpermeabilized cells and viable cells that had been successfully permeabilized and resealed. The accessibility of the cell interior during permeabilization was investigated by including fluorescein isothiocyanate (FITC)-labelled dextrans (11, 38 and 148 kDa) and bovine serum albumin (67 kDa) in the permeabilization buffer, and localizing the FITC label using confocal microscopy following resealing. The confocal images showed that these molecules entered the cells and were retained after resealing. Following the permeabilization-resealing protocol, cells appeared to have both normal morphology and response to electrical stimulation. Thus this appears to be a cheap, simple and effective method to introduce relatively large molecules into cardiac myocytes.
Fawcett
J M
JM
Department of Physiology, University of Leeds, UK.
Harrison
S M
SM
Orchard
C H
CH
D000818:Animals; D001426:Bacterial Proteins; D002417:Cattle / Q000097:blood / Q000196:embryology; D002463:Cell Membrane Permeability / Q000502:physiology; D002469:Cell Separation; D005260:Female; D005312:Fetal Blood / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006352:Heart Ventricles; D008297:Male; D008722:Methods; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats; D017208:Rats, Wistar; D013301:Streptolysins / Q000494:pharmacology; D013997:Time Factors
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D013301:Streptolysins; C025964:streptolysin O; D016650:Fluorescein-5-isothiocyanate