Fast electrical lysis of cells for capillary electrophoresis.
In the past decade, capillary electrophoresis has demonstrated increasing utility for the quantitative analysis of single cells. New applications for the analysis of dynamic cellular properties demand sampling methods with sufficient temporal resolution to accurately measure these processes. In particular, intracellular signaling pathways involving many enzymes can be modulated on subsecond time scales. We have developed a technique to rapidly lyse an adherent mammalian cell using a single electrical pulse followed by efficient loading of the cellular contents into a capillary. Microfabricated electrodes were designed to create a maximum voltage drop across the flattened cell's plasma membrane at a minimum interelectrode voltage. The influence of the interelectrode distance, pulse duration, and pulse strength on the rate of cell lysis was determined. The ability to rapidly lyse a cell and collect and separate the cellular contents was demonstrated by loading cells with Oregon Green and two isomers of carboxyfluorescein. All three fluorophores were detected with a separation efficiency comparable to that of standards. Parallel comparison of electrical lysis to that produced by a laser-based lysis system revealed that the sampling efficiencies of the two techniques were comparable. Rapid cell lysis by an electrical pulse may increase the application of capillary electrophoresis to the study of cellular dynamics requiring fast sampling times.
Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
Futian
F
Han
Yan
Y
Wang
Christopher E
CE
Sims
Mark
M
Bachman
Ruisheng
R
Chang
G P
GP
Li
Nancy L
NL
Allbritton
D000818:Animals; D002458:Cell Fractionation / Q000295:instrumentation* / Q000379:methods*; D045744:Cell Line, Tumor; D004566:Electrodes; D004572:Electrolysis; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins; D007938:Leukemia / Q000473:pathology; D051381:Rats; D013997:Time Factors
D005452:Fluoresceins; C024098:6-carboxyfluorescein