Presentation Title

Innovative Creation of a Laboratory Exercise: Fluorescent Staining of Primary Chicken Embryo Fibroblast Culture Using MitoTracker and Hoechst stains.

Location

Reed 330

Start Date

20-4-2017 7:00 PM

Description

Primary chicken embryo fibroblast cell cultures have been grown in the Developmental Biology course laboratory for many years. With the acquisition of a fluorescent microscope in the Biological Sciences department, it was possible to create a new laboratory exercise incorporating fluorescent staining of these cells. During the fall 2016 semester, primary chicken embryo fibroblast cell cultures were generated from 14 day old chicken embryos, then grown in EMEM with 10% FBS. Living cells were first treated with MitoTracker Orange CMTMRos that stains the mitochondria fluorescent orange. Cells were fixed and permeabilized, then counterstained with Hoechst 33342 DNA stain which stains the nucleus fluorescent blue. Since fluorescent staining of primary chicken embryo fibroblasts has been done on a limited basis in the literature, optimum stain concentrations and incubation times were not readily available. Also, time and funds were not available the summer before this semester to determine optimum parameters. Therefore, during this exercise, student groups tested different concentrations of each reagent to determine optimum staining parameters. Stained cells were visualized on the Nikon inverted fluorescent microscope and pictures were taken. Using class data the optimum MitoTracker and Hoechst stain parameters were determined for future laboratory exercises.

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Apr 20th, 7:00 PM

Innovative Creation of a Laboratory Exercise: Fluorescent Staining of Primary Chicken Embryo Fibroblast Culture Using MitoTracker and Hoechst stains.

Reed 330

Primary chicken embryo fibroblast cell cultures have been grown in the Developmental Biology course laboratory for many years. With the acquisition of a fluorescent microscope in the Biological Sciences department, it was possible to create a new laboratory exercise incorporating fluorescent staining of these cells. During the fall 2016 semester, primary chicken embryo fibroblast cell cultures were generated from 14 day old chicken embryos, then grown in EMEM with 10% FBS. Living cells were first treated with MitoTracker Orange CMTMRos that stains the mitochondria fluorescent orange. Cells were fixed and permeabilized, then counterstained with Hoechst 33342 DNA stain which stains the nucleus fluorescent blue. Since fluorescent staining of primary chicken embryo fibroblasts has been done on a limited basis in the literature, optimum stain concentrations and incubation times were not readily available. Also, time and funds were not available the summer before this semester to determine optimum parameters. Therefore, during this exercise, student groups tested different concentrations of each reagent to determine optimum staining parameters. Stained cells were visualized on the Nikon inverted fluorescent microscope and pictures were taken. Using class data the optimum MitoTracker and Hoechst stain parameters were determined for future laboratory exercises.