Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The overall goal of this procedure is to prepare a single cell suspension from the mouse skin to identify the different populations of resident immune cells via cytofluorimetric analysis. to identify and isolate the different population of skin resident immune cells. Using this technique researchers can easily digest the samples from different regions of the mouse skin without damage to the immune cells surface makers of interest.

To obtain skin samples from the mouse ear cut off the hairless parts of the ears and use forceps to pull apart the dorsal and ventral sides of the organs. Then, gently scrape the inner ear to remove any remaining cartilage. To obtain chunk skin samples, shave all of the hair from the animal with a pair of clippers, followed by the application of depilatory lotion.

After a few minutes clean off the lotion and make a vertical cut in the middle of the mouse back continuing the incision along the borders of the shaved areas around the mouse body. Using forceps to hold the skin in place while separating the sample with the closed, round edged tips of a pair of surgical scissors or a scalpel, gently separate the skin from the peritoneum and back muscles. Then submerge the skin in a 100mm cell culture plate containing 10ml of ice cold PBS and use forceps to scrape off the subcutaneous fat.

To obtain a tail skin sample, cut off the tail and use surgical scissors to make a vertical cut starting from the base of the tail down to the tip. Using forceps, grab the edge of the incision at the base of the tail, then grasping the body of the tail with another pair of forceps, gently pull the skin toward the tip. Remove any excess fat by gently scraping the skin sample.

The foot of the mouse is cut, and then to obtain a skin sample from the foot pad, use surgical scissors to cut the skin of the foot longitudinally from the ankle to the beginning of the digits, taking care to avoid cutting any hair. Then grasp the ankle bones with forceps while grabbing the skin with the other and pull the skin toward the digits as when removing a glove. To label the skin cells, for each sample in turn transfer the tissue into a 35 milimeter petri dish containing 2 milliliters of freshly prepared digestion cocktail.

Using scissors chop the sample into small pieces and incubate the tissue fragments at 37 degrees celsius for 90 minutes with one to two gentle shakes during the incubation period. Next, pour the digested skin tissue onto a 70 micron cell strainer in a 66 milimeter petri dish containing 1 milliliter of RPMI 1640 supplemented with FBS per dish. Wash the strainer with five milliliters of fresh medium then use a syringe plunger to squeeze the samples through the mesh of the strainer.

Rinse the plunger, dish, and strainer with 20 milliliters of fresh medium and transfer the tissue slurry into a 50 milliliter conical tube. After all of the samples have been processed centrifuge the cells and re-suspend the pellets in 100 microliters per sample of two micrograms per milliliter of anti CD16 CD32 blocking antibody in PBS plus 1%FBS. After 15 minutes at four degrees celsius add 100 microliters of antibody as indicated in the table to the appropriate samples for 30 minutes at four degrees celsius protected from light.

At the end of the incubation wash the cells in one milliliter of PBS plus FBS and re-suspend the pellets in 300 microliters of FBS PBS. Then label the cells with 2 micrograms per milliliter of DAPI for 10 minutes at four degrees celsius protected from light. To isolate the leukocytes, first gate the DAPI positive population to eliminate the dead cells.

Then select the singlets in a forward scatter area by forward scatter width plot and gate the cells based on the size and granularity of the leukocytes of interest. Next, select the CD45 positive cells, and then for each sample identify the skin dendritic cell populations by their CD11C and MHC class two expression. Identify the macrophages by their CD64 and F480 expression the B cells by their CD19 and MHC class two expression and the T cells by the CD4 or CD8 expression.

The isolation of skin immune cells as just demonstrated yields a population of more than 50%viable cells from all regions of the skin, between 5, 000 and 15, 000 per square centimeter of which are CD45 positive. A sufficient number of dendritic cells, macrophages, B cells, and T cells are also obtainable for downstream analysis from one square centimeter of mouse skin, as indicated. After watching this video you should have a good understanding of how to digest a non inflamed skin tissue and to isolate the leukocytes for fax analysis.