Delivery of macromolecules into adherent cells via electroporation for use in fluorescence spectroscopic imaging and metabolic studies.
A method is described to introduce by electroporation membrane-impermeant molecules into adherent living cells with little perturbation. The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based 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 microF capacitance at 4 degrees C is a good starting point for many cell types. Electrode distance from the adherent cells was critical at 1.0 +/- 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 sucrose. 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.
Bright
G R
GR
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4930, USA. grb4@cwru.edu
Kuo
N T
NT
Chow
D
D
Burden
S
S
Dowe
C
C
Przybylski
R J
RJ
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival; D003847:Deoxyglucose / Q000378:metabolism; D004553:Electric Conductivity; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D051272:Glucose Transporter Type 1; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D016716:PC12 Cells; D019000:Phosphotyrosine / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D051272:Glucose Transporter Type 1; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D003847:Deoxyglucose