Perform the protocol inside a biosafety cabinet on the euthanized mouse appropriately shaved and disinfected with 70%ethanol. Create a 0.5-inch superficial incision in the animal's abdomen using scissors. Cut longitudinally along the midline towards the neck and lower abdomen.
Additionally, make lateral cuts to flap open the skin. Using blunt forceps, gently separate the skin from surrounding tissues, while going around the animal's torso cutting the skin laterally around the neck and the lower abdomen. Carefully place the skin with the dermis side facing down in a 100-millimeter tissue culture plate.
And ensure the skin is as stretched as possible. Add six milliliters of Dispase solution to the plate, ensuring the skin is completely covered. Then, cover the plate.
Wrap the plate with Parafilm. Incubate the plate at room temperature for 10 minutes to flatten the explant. The next day, using a pipette, remove the liquid from the plate containing the isolated skin.
Then, with the aid of forceps, separate the dermis from the epidermis while removing any visible adipose tissue from it. And place the isolated dermis into a clean tissue culture plate. Add one milliliter of DMEM containing 1X antibiotic-antimycotic solution to the dermis in the culture plate.
Tilt the plate to accumulate the liquid on one side of the plate. Then, mince the dermis into pieces of one to two square millimeters. And transfer them to a new 50-milliliter conical tube.
Add 10 milliliters of collagenase type II solution to the tube. Incubate it for two hours at 37 degrees Celsius with continuous agitation at 30 to 50 rpm to digest the dermis. Filter the cell suspension through a 70-micron cell strainer.
And centrifuge it at 250g for five minutes. After discarding the supernatant, resuspend the pellet in 10 millimeters of DMEM containing antibiotic-antimycotic solution. Filter the cell suspension through a 40-micron cell strainer before centrifuging, as demonstrated previously.
Resuspend the resulting cell pellet in one milliliter of complete LEC media. Then, plate the cells in complete LEC media in a collagen-coated 60-millimeter tissue culture plate. Replace the media every two days by washing cells with PBS twice and adding fresh LEC media.
Low magnification images of cells isolated from the murine dermis showed a mixed cell population.
