title: Electroporation of adherent cells with low sample volumes on a microscope stage.
abstract: The labeling of specific molecules and their artificial control in living cells are powerful techniques for investigating intracellular molecular dynamics. To use these techniques, molecular compounds (hereinafter described simply as 'samples') need to be loaded into cells. Electroporation techniques are exploited to load membrane-impermeant samples into cells. Here, we developed a new electroporator with four special characteristics. (1) Electric pulses are applied to the adherent cells directly, without removing them from the substratum. (2) Samples can be loaded into the adherent cells while observing them on the stage of an inverted microscope. (3) Only 2 μl of sample solution is sufficient. (4) The device is very easy to use, as the cuvette, which is connected to the tip of a commercially available auto-pipette, is manipulated by hand. Using our device, we loaded a fluorescent probe of actin filaments, Alexa Fluor 546 phalloidin, into migrating keratocytes. The level of this probe in the cells could be easily adjusted by changing its concentration in the electroporation medium. Samples could be loaded into keratocytes, neutrophil-like HL-60 cells and Dictyostelium cells on a coverslip, and keratocytes on an elastic silicone substratum. The new device should be useful for a wide range of adherent cells and allow electroporation for cells on various types of the substrata.
authors.affiliation: Department of Functional Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8512, Japan.
authors.forename: Harunobu
authors.initials: H
authors.lastname: Tsugiyama
authors.forename: Chika
authors.initials: C
authors.lastname: Okimura
authors.forename: Takafumi
authors.initials: T
authors.lastname: Mizuno
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Iwadate
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000818:Animals; D002465:Cell Movement; D002478:Cells, Cultured; D004023:Dictyostelium / Q000166:cytology; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000008:administration & dosage* / Q000032:analysis; D006054:Goldfish; D018922:HL-60 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds / Q000008:administration & dosage* / Q000032:analysis; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods
chemical_list: C481052:Alexa fluor 546; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds
keywords: Dictyostelium cells; HL-60 cells; cell migration; keratocytes; speckle microscopy