Localized sampling of cytoplasm from Xenopus oocytes for capillary electrophoresis.
Continued progress in cellular physiology requires new measurement strategies which can be applied to solitary cells. Since many cellular signaling pathways act on time scales of a few seconds, there is a critical need for single-cell techniques with subsecond time resolution. Capillary electrophoresis shows great promise as a tool for the analysis of individual cells. In the present work, we describe a technique to load a capillary with picoliter to nanoliter volumes of cytoplasm and initiate electrophoresis in less than 500 ms. When cytoplasm was sampled from a Xenopus laevis oocyte previously loaded with fluorescein, calcium green, or a mixture of the two fluorophores, their fluorescent peaks were readily identifiable on the electropherogram. Since the volume of cytoplasm (< or = 30 nL) loaded into the capillary was much smaller than the 1 microL oocyte volume, spatially localized biochemical measurements were also possible. To demonstrate the utility of this new technique, the activity of the enzyme beta-galactosidase was measured in small regions of the Xenopus oocyte. Subcellular, subsecond sampling of oocyte cytoplasm will enable biochemical measurements with the resolution required to understand many cellular signal transduction pathways.
Luzzi
V
V
Department of Physiology and Biophysics, University of California, Irvine 92697-4560, USA.
Lee
C L
CL
Allbritton
N L
NL
D000818:Animals; D003593:Cytoplasm / Q000737:chemistry* / Q000201:enzymology; D019075:Electrophoresis, Capillary; D009865:Oocytes / Q000737:chemistry* / Q000201:enzymology; D012313:RNA / Q000096:biosynthesis; D014981:Xenopus; D001616:beta-Galactosidase / Q000032:analysis
D012313:RNA; D001616:beta-Galactosidase