Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

Begin by using surgical scissors to harvest the tongue from a 6-week-old male or female B6 transgenic mouse. Use a scalpel to mince the tissue into very small fragments and collect the pieces into a 15-milliliter tube containing 4.5 milliliters of RPMI plain medium without serum. Add 200 microliters of triple enzyme mix to the tube and incubate the sample at 37 degrees Celsius for 30 minutes, tapping the tube every 10 minutes to enhance the enzymatic dissociation of the tissue. At the end of the incubation, stop the reaction with 5% FBS in PBS, and filter the cell suspension through a 70-micrometer mesh strainer to separate the cells from the larger tissue fragments.

Collect the cells by centrifugation and resuspend the pellet in 3 milliliters of complete medium. Then, plate the cells in 3 milliliters of complete medium per 60-millimeter dish. Transfer cell aggregates retained on top of the filter to a separate 60-millimeter culture dish and add 3 milliliters of complete medium. Keep both dishes in the incubator until distinct cell colonies are formed.