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Unformatted text preview: Polina Pomerants 3096 Lab Naoto Tanaka Room 235 Cell Culture and Fluorescence Objective: The purpose of the cell culture experiment was to first dissociate cells from the body tissues of a chick embryo and to grow these cells in vitro in primary culture. The aim of the fluorescence experiment was to stain both chick fibroblasts and buccal (cheek) cells with a fluorescence stain reagent, consisting of three dyes - wheat germ agglutinin (WGA), concanavalin A (Con A), and 4'-6-diamidino-2-phenylindole, dihydrochloride (DAPI). Furthermore, the aim of the experiment was to view slides of these stained cells under the fluorescence microscope, and compare the staining effects achieved by the three dyes between images of fibroblasts and images of cheek cells, analyze differences between the color of illuminating light and the color of fluorescence among the three stains, and also contrast the appearance of fluorescence images against the phase contrast microscope-generated images. Methods: In the cell culture experiment, an embryo tissue was isolated from a 6-day old egg through the airspace using sterile techniques. Sterilization was critical in order to prevent bacterial contamination during embryo tissue dissociation. To expose the chorioallantois, the eggshell was punctured inside the diameter of the airspace and an opening was made around the circle. The chorioallantoic membrane, separating the airspace from the embryonic fluid, was removed to disclose the embryo. The embryo was then extracted and immersed in 4 ml of - 1 - phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic; pH 7.4) (1). The PBS, a buffer used to maintain the osmolarity of the cells to keep the cells in an isotonic state, washed the embryo to clear away any residual membrane parts. The head, limbs, and viscera were removed from the embryo, and the remaining embryo was again washed with 4 ml of PBS to get rid of any residual chick body contents. To dissociate the tissue, the embryo tissue was then minced into grain sized pieces, after which the minced tissue was resuspended in 4ml of PBS solution. The tissue pieces were then allowed to settle and the PBS-containing supernatant removed. This process of tissue resuspension, tissue stirring, tissue settling, and supernatant removal was repeated twice in order to wash away residual tissue extracts. PBS, which contains no Ca2+/Mg2+, was used to wash the cells prior to trypsinisation in order to reduce the concentration of divalent cations and proteins that inhibit trypsin action. The minced and resuspended tissue was then treated with and incubated for 20 minutes at 37 C in cold sterilized 4 ml of 10 mg/ml trypsin, a serine protease which degrades the extracellular matrix that covers the cells (1). Following incubation, the trypsin solution was removed from the tissue and the residual trypsin in the resulting tissue pellet was then neutralized with two resuspensions in 4 ml of PBS. To remove the residual trypsin was then neutralized with two resuspensions in 4 ml of PBS....
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- Fall '09