Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

The overall goal of this protocol is to obtain single-cell suspensions from the murine aorta, kidney, and lymph nodes to quantify and characterize immune cells in a model of angiotensin II-induced hypertension. This method can help answer key questions in the field of cardiovascular disease, such as which two lymphocyte subsets promote the development of hypertension. The main advantage of this technique is that it allows the identification and quantification of immune cell subsets in solid organs at the single-cell level.

Before beginning the procedure, disinfect the mouse chest with 70%ethanol. Then use scissors to carefully open the skin and chest wall to expose the heart. To perfuse the vasculature, make a small incision in the right atrium and steadily inject at least 10 milliliters of cold PBS at a rate of approximately one milliliter per second into the apex of the left ventricle.

When all of the organs have blanched, use fine-dissecting forceps to remove the abdominal aortic-draining lymph nodes, taking care not to rupture the capsules. Under a dissecting microscope, remove the two kidneys with fine scissors. Then use fine curved scissors to dissect the entire thoracic and abdominal aorta starting at the heart and ending at the iliac bifurcation, taking care to keep the surrounding perivascular fat attached to the aorta.

Next, transfer the kidneys into a dissociation tube containing 10 milliliters of kidney digestion solution and use a semi-automated dissociator to mechanically disaggregate the kidney tissue according to the manufacturer's instructions. At the end of the dissociation, incubate the samples at 37 degrees Celsius under continuous rotation. After 20 minutes, immediately stop the enzymatic digestion with cold RPMI 1640 medium supplemented with 5%FBS and filter the resulting tissue slurry through a 40-micron strainer into a 50-milliliter conical tube.

Use the plunger from a one-milliliter syringe to tease apart the remaining tissue. Then pellet the cells by centrifugation. Resuspend the pellet in three milliliters of 36%density gradient medium and transfer the cells to a 15-milliliter conical tube.

Gently layer three milliliters of 72%density gradient medium under the cells, taking care not to disturb the layers, and separate the cells by centrifugation. The immune cells will be located at the interface. Remove the topmost yellowish layer containing cellular debris and then bring the volume up to 15 milliliters using cold PBS.

Shake well to break down the gradient and then wash the cells by centrifugation. To isolate the aortic immune cells, transfer the aorta into a two-milliliter tube containing one milliliter of freshly prepared aorta-digestion solution on ice and use fine scissors to mince the whole aorta into a fine pieces. Then incubate the fragments at 37 degrees Celsius for 30 minutes of continuous rotation.

At the end of the digestion, add cold RPMI supplemented with 5%FBS at five times the volume of digestion solution and filter the resulting tissue slurry through a 40-micron strainer. Then collect the single-cell suspension by centrifugation and resuspend the pellet in fresh RPMI with FBS. To generate a single-cell suspension from the aortic-draining lymph nodes, transfer the lymph nodes onto a 40-micron strainer in a 60-by-15 millimeter tissue culture dish and use a one-milliliter syringe plunger to disaggregate the tissue.

Then flush the strainer with two milliliters of RPMI plus FBS and transfer the liberated cells into a 15-milliliter conical tube. To stimulate the cells, first adjust each cell suspension to a one times 10 to the sixth cells per milliliter concentration in cell culture medium. Then add two microliters of the cell activation cocktail to each group of cells for every one milliliter of medium.

Next, transfer one to two times 10 to the sixth aortic or kidney cells per well into a 12-well plate and one to two times 10 to the sixth lymph node cells per well into a 24-well plate. Place the plates in a 37 degree Celsius humidified carbon dioxide incubator for three hours. Then transfer the cells into the appropriate corresponding polystyrene FACS tubes and pellet the cells by centrifugation.

After washing the cells two times with fresh FACS buffer under the same centrifuge conditions, block and non-specific surface binding with FC block and wash the cells in PBS. Resuspend the pellets in one milliliter of fresh PBS and stain the cells with one microliter of a cell-impermeant amine-reactive viability stain for 15 minutes at four degrees Celsius, protected from light. At the end of the incubation, wash the cells two more times in one to two milliliters of FACS buffer and resuspend the pellets in 100 microliters of the appropriate cell-surface staining antibody cocktail.

After 30 minutes at four degrees Celsius and protected from light, wash the cells two more times in two milliliters of fresh FACS buffer, and fix and permeabilize the cells with a fixation and permeabilization kit according to the manufacturer's instructions. Next, label the cells with the intracellular antibody cocktail of interest and wash the cells in FACS buffer again. For flow cytometric analysis of the stimulated intracellular cytokine production, first run the unstained and single-stained control tubes to adjust for the spectral overlap, followed by the fluorescence-minus-one controls for each fluorophore in the panel.

When the basic fluorescence analysis parameters have been set, use a forward-by-side scatter area plot to set the primary gate for the leukocyte population detection, followed by gating to exclude the dead cells according to their lack of viability-stain expression. Plot the forward scatter area against the forward scatter height and gate to exclude the doublets. The leukocytes can then be identified by their CD45 expression.

To determine the presence of IL-17A or IL-17F producing T-cells with the murine kidney or aorta, single-cell suspensions can be prepared as just demonstrated for fluorescence-minus-one control analysis of each cytokine. As expected, very few cells are positive for IL-17A or IL-17F in the fluorescence-minus-one controls, permitting the accurate discrimination of positive versus negative signals to properly adjust the gates for the identification of IL-17A or IL-17F positive cells in the experimental samples. Representative intracellular staining of T-cells isolated from the kidney and the aorta of wild-type mice infused with vehicle or angiotensin II reveals the presence of a subset of IL-17A or IL-17F-expressing T-cells within both tissues.

Further, the intracellular staining of T-cells from the aortic-draining lymph nodes demonstrates that CD8 positive T-cells express interferon gamma and T-bet following angiotensin II stimulation. Once mastered, this technique can be performed in six to nine hours depending upon the number of tissues being processed. While performing this procedure, it is important to work quickly and efficiently to minimize cell death.

After watching this video, you should have a good understanding of how to generate single-cell suspension from the murine kidney, aorta, and lymph nodes and to characterize and quantify immune cell subsets from these organs. This technique allows cardiovascular and renal physiologies to explore the changes in immune cell populations that may contribute to hypertension and other pro-inflammatory diseases.