The overall goal of this procedure is to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient separation. This method can be applied to the investigation of cellular and molecular mechanisms in many mouse models of skin pathologies, including atopic dermatitis. Though this method can provide insight into skin-infiltrating leukocyte biology, it can also be applied to the investigation of other resident cell populations.

Generally, individuals new to this method will struggle with the skin harvesting, which includes a complete removal of the excess adipose and connective tissues. Visual demonstration of this method is critical, as skin harvesting and density gradient handling are difficult to learn, and require precision and practice. At the appropriate time point after treatment, use 10 centimeter surgical scissors to carefully excise a 25 by 25 millimeter area of shaved flank skin from an adult eight to twelve-week-old female ND4 Swiss-Webster mouse.

Use a clean, sharp surgical blade to scrape away the excess fat and connective tissue from the skin, then transfer the skin into a 50 milliliter conical tube containing 10 milliliters of room temperature HBSS supplemented with EDTA, HEPES, and FBS, and predigest the tissue on a rotating plate for 30 minutes in a dry, non-gassed, 37 degree Celsius incubator. At the end of the incubation, vortex the tube vigorously for ten seconds and filter the tissue slurry through a 70 micron strainer. Next, use a pair of forceps to transfer the flank skin pieces from the strainer into a new 50 milliliter tube containing ten milliliters of fresh, room temperature HBSS media, supplemented with EDTA, HEPES, and FBS, then use a 12.5 centimeter pair of surgical dissecting scissors to mince each piece of flank skin so that the average piece of tissue is approximately 2.2 by 2.2 millimeters in size.

When all of the pieces have been minced, return the tube to the rotating plate for another 30 minute incubation. Vortex the tube vigorously for 10 seconds at the end of the incubation and filter the contents through the 70 micron strainer. Collect the remaining flank skin pieces in a new 50 milliliter tube containing 10 milliliters of HBSS supplemented with 0.7 milligrams per milliliter of Collagenase D, and return the tube to the rotating plate for another 30 minute incubation.

After another 10 seconds of vortexing, filter the contents of the tube again, this time discarding the debris on the strainer, then fill the tube with up to 50 milliliters of HBSS, EDTA, HEPES, FBS, spin down the cells and decant the supernatant. Add three milliliters of room temperature, 67 percent density gradient centrifugation medium in PBS to a new 15 milliliter conical tube, and resuspend the cell pellet in five milliliters of room temperature 44 percent density gradient centrifugation medium in HBSS with phenol red. Setting the pipettor to the lowest speed, place the pipette on the side of the tube and gently layer the cells on top of the 67 percent density gradient centrifugation medium.

When working with density gradients, take care to avoid making bubbles and to minimize any disruptions to the gradient interface. Then separate the cells, using a five milliliter transfer pipette to collect the cells at the interface at the end of the centrifugation. As gently layering the gradient and carefully extracting the interface of the gradients are critical steps, it is wise to practice the density gradient centrifugation medium layering and interface removal with the suspension of murine splenocytes before attempting the procedure with the experimental cells of interest.

Finally, wash the collected cells in ice-cold PBS supplemented with FBS in a 15 milliliter conical tube and count the number of viable cells by trypan blue exclusion. CD4 and CD8-alpha surface expression are not affected by Collagenase D digestion, as the same number of CD4 positive and CD8-alpha positive cells is detected with or without enzymatic digestion. Further, Collagenase D digestion exhibits no effect on the surface density of the T cell activation markers CD44 and CD62L on CD4 positive T cells.

After gradient separation, about 250 viable immune cells per square millimeter of flank tissue are isolated from Oxazolone, or Ox-challenged mice, versus only four viable immune cells per square millimeter of tissue in vehicle-challenged mice, previously sensitized with Ox.95 percent of the low forward and side scatter single cells in Ox-challenged mice, and 71 percent of these cells in vehicle-challenged controls are live CD45 positive cells, equivalent to an approximately 67 fold increase in the infiltrating CD45 positive immune cell population in the flank skin following three daily Ox challenges. Indeed, Ox challenge results in the pronounced infiltration of T cells and neutrophils into the flank skin, with 42 percent of the single, live CD45 positive cells in Ox-treated animals, apparent Gr-1 positive neutrophils, and approximately 84 percent CD3 positive T cells expressing CD8-alpha, or CD4, compared to control, ethanol-treated animals. Once mastered, this technique can be completed in about three hours if it's performed properly.

While attempting this procedure, it's important to remember to handle the density gradient carefully. Don't forget to always use room temperature density gradient centrifugation medium when performing this procedure. Following this procedure, other methods like flow cytometric analysis, cell sorting, cell transfer, or in vitro cell culture can be performed to evaluate the phenotype and function of the skin-infiltrating leukocytes.

After watching this video, you should have a good understanding of how to isolate skin-infiltrating leukocytes using enzymatic digestion and density gradient centrifugation.