An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

This method can help answer questions in the immunometabolism field about the contribution of immune cells to obesity-induced inflammation in adipose and steatohepatitis in hepatic tissue. These techniques have been optimized for the isolation of adipose and hepatic immune cells in a diet-induced model of nonalcoholic steatohepatitis achieved through a non-specific pathogen-free rodent housing. These methods can help to deepen our understanding of the immunological mechanisms involved in the maintenance of metabolic homeostasis.

Generally, individuals new to this technique will struggle with maximizing the yield of functionally viable dissociated cells and setting up the proper gating in flow cytometry. To generate an adipose tissue single-cell suspension, spray the chest of a euthanized mouse with 70%ethanol, and carefully make a five to six-centimeter central incision through the integument and abdominal wall along the entire length of the rib cage to expose the pleural cavity and heart. Using a 26-gauge needle, inject at least 10 milliliters of 0.9%saline solution into the apex of the left ventricle, and use scissors to open the peritoneal cavity.

Then, dissect the epigonadal adipose tissue, and weigh it. Mechanically dissociate the adipose tissue into fine pieces in a Petri dish at four degrees Celsius before transferring the tissue fragments into a 50-milliliter conical tube. Rinse the Petri dish with one milliliter of 0.5%bovine serum albumin, or BSA, in PBS, and add three milliliters of freshly prepared adipose tissue digest solution per gram of adipose tissue to the tube.

After 20 minutes at 37 degrees Celsius and 200 rotations per minute, add five milliliters of 0.5%BSA in PBS, and place the adipose digest on ice. Triturate the solution multiple times with a 10-milliliter serological pipette, and use a plunger to pass the solution through a 100-micrometer strainer. Separate the tissue fractions by centrifugation, and use a pipette to discard the floating adipocyte fraction.

Resuspend the stromal vascular fraction cell pellet in one milliliter of ammonium-chloride-potassium, or ACK, lysis buffer, stopping the lysis reaction with 10 milliliters of 2%fetal calf serum, or FCS, in PBS after a few seconds. Then, collect the isolated white blood cells by centrifugation, and resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. Harvest the liver into a conical tube of PBS on ice before using syringe stamps to mechanically dissociate the tissue in a Petri dish at four degrees Celsius.

Transfer the dissected liver tissue fragments into a 50-milliliter tube containing 10 milliliters of warm liver digest solution, and rinse the Petri dish with three milliliters of fresh liver digest solution. After 20 minutes at 37 degrees Celsius and 200 rotations per minute, stop the digestion with 20 milliliters of HBSS, and triturate the tissue slurry multiple times with a 10-milliliter serological pipette. Use a plunger to pass the tissue solution through a 100-micrometer strainer, and collect the liver cells by centrifugation.

Resuspend the pellet in 20 milliliters of fresh HBSS, and remove the hepatocyte matrix by centrifugation. Collect the supernatant for centrifugation, resuspending the pellet in 10 milliliters of 33%low viscosity density gradient medium solution in HBSS. Separate the liver immune cells by density gradient centrifugation, and resuspend the leukocyte pellet in one milliliter of ACK lysis buffer.

After four minutes at room temperature, stop the lysis with 10 milliliters of HBSS, and filter the cells through a 30-micrometer pore strainer into a 15-milliliter tube for centrifugation. Then, resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. For flow cytometric analysis of the isolated leukocyte populations, add up to three times 10 to the six cells in 100 microliters of 2%FCS in PBS from the tissue of interest into individual polystyrene fluorescence-activated cell sorting, or FACS, tubes.

Add 10 microliters of anti-CD16/CD32 antibody to each tube to block any non-specific binding for 10 minutes on ice, followed by staining with the appropriate volume of antibody mixture and one microliter of viability dye per sample to allow live and dead cell discrimination. After 20 minutes at four degrees Celsius protected from light, wash the samples two times with two milliliters of 2%FCS plus PBS per wash, and centrifuge at 300 times g for five minutes at four degrees Celsius. Then, resuspend the pellet in fresh PBS plus FCS.

To analyze the cells by flow cytometry, use an unstained negative control sample to set the forward and side scatter, and adjust the voltages of the flow cytometer to detect the viable leukocyte population and to exclude the debris. Next, run single stained control samples for multicolor compensation before running the experimental samples, collecting at least five times 10 to the four events per tube. Then, export the FCS data files for analysis, and set the gating strategy for CD45-positive leukocytes to allow the identification of the subsequent immune cell populations of interest.

Here, a representative gating strategy for T cell subpopulations including doublet discrimination and viability staining in murine perigonadal fat is shown. CD45-positive leukocytes are first gated for CD4 and CD8 and subsequently for CD44 and CD62 ligand to discriminate between naive, central memory, effector memory, and effector T cells. CD44-positive cells are then further characterized with CD127, killer cell lectin-like receptor G1, and programmed death-1.

Here, the gating strategy for analyzing B cells, granulocytes, natural killer cells, macrophages, and dendritic cells is shown. A higher percentage of effector memory CD4-positive and CD8-positive cells can be detected in high-fat diet mice housed in non-SPF conditions, whereas intrahepatic naive CD4-positive and CD8-positive T cells are found to be considerably lower in these animals compared to SPF high-fat diet mice at week seven. Severe steatosis, including an increase of large lipid droplets resulting in macrovesicular steatosis, lobular inflammation, hepatocellular ballooning, and destroyed lobular structure is found in high-fat diet exposed mice while only some SPF mice display a mild fat accumulation in the liver.

In addition, the percentage of natural killer cells is higher in the perigonadal adipose tissue of high-fat diet exposed mice, whereas high-fat diet SPF mice demonstrated higher percentages of dendritic cells. When attempting this procedure, it is important to keep the cells on ice and in dark unless otherwise noted. You should now have a good understanding of how to properly prepare single-cell suspensions and how to isolate murine adipose and hepatic tissue for flow cytometry experiments.