The overall goal of this protocol is to isolate different populations of stem cells and epidermal keratinocytes from the dorsal mouse skin. This protocol allows the user to simultaneously isolate multiple cell populations from the dorsal mouse skin. The main advantages of this technique are that it is fast, reliable, and can be used to isolate specific cell types of interest from a mixture of heterogeneous skin cell populations.
Demonstrating the procedure will be Dr.Yahav Yosefzon from my laboratory. Begin by using electric clippers to shave all of the dorsal skin hair from a 50 to 80 day old mouse in the telogen phase of the hair follicle cycle, keeping the clippers as close to the skin as possible, without damaging the skin and subcutaneous layer. Next, use 70%ethanol to disinfect the skin and to remove any hair residue.
Place the mouse onto a dissecting pad. Then use forceps to lift the skin near the tail, and nick the skin with scissors. To harvest the entire dorsal skin, carefully cut the tissue in a posterior-anterior direction, separating the skin from the fascia.
When all of the skin has been removed, pin the two adjacent edges of the tissue onto a dissecting mat, hair side down. Then, using curved forceps, apply light pressure to keep the skin from tearing and gently scrape away the fat with a blunted scalpel until the dermis is clear and neat. When all of the fat has been removed, place the skin dermis side down in a 100 millimeter sterile culture dish, and straighten out the tissue until there are no folds.
Wash the skin with 10 milliliters of PBS without calcium and magnesium, straightening the skin again as necessary. Then remove the PBS and incubate the tissue in 10 milliliters of 0.25%trypsin, taking care that the skin is unfolded and freely floating. At the end of the incubation, using curved forceps to hold the skin in place, scrape all of the hair from the skin, starting at the tail and following the direction of hair growth.
As the follicles tend to form small clumps, scrape small areas at a time to reduce the size of the clumps as much as possible. When all of the hair follicles have been collected, transfer the hairless skin to a new culture dish and hydrate the tissue with 10 milliliters of PBS without calcium and magnesium. Confirm that all of the hair follicles have been removed.
Then use a scalpel and forceps to break down the follicles until a single hair follicle suspension is obtained. Next, vigorously triturate the hair follicle solution with a 10 milliliter pipette for a few minutes to break up all of the clumps. Then transfer the cells into 50 milliliter tubes on ice, pre-labeled with the animal and sample numbers.
Now aspirate the PBS from the skin and use it to rinse the cell culture dish that contained the hair follicle suspension. Pool the wash with the hair follicle cells and filter the cell suspension through a 70 micron cell strainer into a new 50 milliliter tube. Wash the first 50 milliliter tube and the strainer with 10 milliliters of staining buffer.
Then filter the suspension through a 40 micron strainer into a new 50 milliliter tube, and wash the second tube with five to 10 milliliters of fresh staining buffer. Next, spin down the cells two times, washing the cells in five milliliters of fresh straining buffer during the second centrifugation. After the second spin, re-suspend the pellet in 800 microliters of fresh staining buffer.
Then transfer 25 to 50 microliters of cells to the unstained and DAPI control tubes, and add enough staining buffer to bring the total volume in each control tube up to 300 microliters. Now add the primary antibodies and DAPI to the appropriate sample tubes and mix the cells with gentle flicking. Label the cells for 30 minutes on ice in the dark with flicking every 10 minutes to keep the cells in suspension.
At the end of the incubation, wash the cells with approximately four milliliters of staining buffer per tube, and re-suspend the pellets in 800 microliters of staining buffer. Then filter the cells into fax tubes with cell strainer caps to ensure a single-cell suspension during their analysis. In these graphs, different patterns of CD34 and Sca-1 cell surface expression within the alpha six positive beta one positive population of skin epithelial cells are shown.
The cells were first gated according to their expression of integrin alpha six and integrin beta one. The integrin alpha six positive integrin beta one positive cells were then gated according to their CD34 and Sca-1 expression. Two distinct populations of cells were observed.
The alpha six beta one CD34 positive Sca-1 negative cells, which represent the hair follicle stem cells, and the alpha six beta one Sca-1 positive CD34 negative cells, which represent the epidermal keratinocytes. Typically, 1.5 to five times 10 to the fifth alpha six beta one positive cells per animal or CD34 positive hair follicle stem cells. Once mastered, this technique can be completed in approximately eight hours, if properly performed.
After watching this video, you should have a good understanding of how to dissociate the mouse dorsal skin and to isolate hair follicle stem cells and epidermal keratinocytes.
