Primary culture of Caenorhabditis elegans developing embryo cells for electrophysiological, cell biological and molecular studies.
Cell culture is an invaluable tool for investigation of basic biological processes. However, technical hurdles including low cell yield, poor cell differentiation and poor attachment to the growth substrate have limited the use of this tool for studies of the genetic model organism Caenorhabditis elegans. This protocol describes a method for the large-scale culture of C. elegans embryo cells. We also describe methods for in vitro RNA interference, fluorescence-activated cell sorting of embryo cells and imaging of cultured cells for patch-clamp electrophysiology studies. Developing embryos are isolated from gravid adult worms. After eggshell removal by enzymatic digestion, embryo cells are dissociated and plated onto glass substrates. Isolated cells terminally differentiate within 24 h. Analysis of gene expression patterns and cell-type frequency suggests that in vitro embryo cell cultures recapitulate the developmental characteristics of L1 larvae. Cultured embryo cells are well suited for physiological analysis as well as molecular and cell biological studies. The embryo cell isolation protocol can be completed in 5-6 h.
Strange
Kevin
K
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. kevin.strange@vanderbilt.edu
Christensen
Michael
M
Morrison
Rebecca
R
D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology*; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D004553:Electric Conductivity; D004625:Embryo, Nonmammalian / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis; D040542:Mechanotransduction, Cellular; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D009474:Neurons / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D034622:RNA Interference; D000071737:Stromal Interaction Molecule 1
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; D000071737:Stromal Interaction Molecule 1; C516629:stim-1 protein, C elegans; D049452:Green Fluorescent Proteins