The number of molecules taken up by electroporated cells: quantitative determination.
Fluorescent and fluorescent-labeled molecules were used with calibrated flow cytometric fluorescence measurements of electrically pulsed cells (intact yeast: Saccharomyces cerevisiae) to demonstrate a method for determining the net number of molecules transported into electroporated cells. For the conditions used, a single pulse of width 50 microseconds and magnitude 8.0 +/- 0.5 kV/cm resulted in an average net molecular uptake which is large, n = 1.4 x 10(5) molecules of 70 kDa FITC-dextran (supplied extracellular concentration of 500 microM), and n = 1.0 x 10(8) molecules of 660 Da propidium iodide (PI; 80 microM). Both molecules were present in pulsed cells at less than equilibrium values, consistent with a transient uptake mechanism. Intracellular FITC-dextran is present in soluble form, while PI is predominantly bound to nucleic acids. A broad, statistically significant distribution of molecular uptake was also observed. Such quantitative determinations should be important for guiding applications of electroporation, and for testing models of electroporation mechanisms. Further, the use of PI, which is well established as a membrane exclusion dye, provides additional support for the interpretation that both PI and FITC-dextran were internalized as a result of an electrical pulse.
Bartoletti
D C
DC
Harvard-M.I.T. Division of Health Sciences and Technology, Cambridge, 02139.
Harrison
G I
GI
Weaver
J C
JC
D001665:Binding Sites; D001692:Biological Transport; D002452:Cell Count; D002463:Cell Membrane Permeability; D004247:DNA / Q000378:metabolism; D003911:Dextrans / Q000032:analysis*; D004553:Electric Conductivity; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000032:analysis*; D010617:Phenanthridines / Q000032:analysis*; D011419:Propidium / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D003911:Dextrans; D005452:Fluoresceins; D010617:Phenanthridines; C015219:fluorescein isothiocyanate dextran; D011419:Propidium; D004247:DNA; D016650:Fluorescein-5-isothiocyanate