title: Measurement of volume injected into individual cells by quantitative fluorescence microscopy.
abstract: Pressure microinjection is frequently used to introduce substances into mammalian cells, but precise quantitation of the volume injected into individual cells has been difficult. A simple and reliable procedure for determining the volume injected was developed in order to determine what intracellular concentration of AMP-PNP was necessary to inhibit specific cellular processes. The technique uses fluorescent Lucifer Yellow-labeled dextrans in the microinjection buffer and quantitative fluorescence microscopy to measure the fluorescence intensity of the injected cell. The volume injected is computed from a standard curve derived from the volume and fluorescence of spherical, microscopic droplets of Lucifer Yellow dextran solution. The droplets are ejected from a micropipet into immersion oil where they sink to rest on a siliconized coverslip. For the measurement of fluorescence, an inexpensive photomultiplier system that is attached to a fluorescence microscope is described. The potential uses of this method for other microassays are discussed.
authors: Department of Zoology, Duke University, Durham, NC 27706.
authors: G M
authors: GM
authors: Lee
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000008:administration & dosage / Q000032:analysis; D000818:Animals; D002460:Cell Line; D003911:Dextrans; D005456:Fluorescent Dyes; D007546:Isoquinolines; D008845:Microinjections / Q000379:methods*; D008856:Microscopy, Fluorescence; D011312:Pressure
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D007546:Isoquinolines; D000266:Adenylyl Imidodiphosphate; C017475:lucifer yellow