Microdissection tools.

Surgical microscope (e.g., Diagnostic Instruments Inc., Sterling Heights, Michigan).

1× Phosphate-buffered saline (PBS): 2.7 mM potassium chloride, 1.5 mM potassium phosphate monobasic, 137.9 mM sodium chloride, 8.1 mM sodium phosphate dibasic.

2 % heparin diluted in PBS.

12 × 75 mm, 5 mL polystyrene round-bottom test tube (FACS tubes).

Collagenase, Type I (e.g., Worthington Biochemicals, Lakewood, New Jersey).

Collagenase, Type XI (e.g., Sigma Aldrich, St. Louis, Missouri).

Deoxyribonuclease I (DNase) from bovine pancreas, type II (e.g., Sigma Aldrich).

Hyaluronidase from bovine testes, type 1-s (e.g., Sigma Aldrich).

Water bath.

70 μm nylon cell strainer.

Syringe without needle.

Table-top, swinging bucket centrifuge 5810R.

Red blood cell lysis buffer: 155 mM ammonium chloride (NH₄Cl), 10 mM potassium bicarbonate (KHCO₃), and 1.27 mM ethylenediaminetetraacetic acid (EDTA) diluted in distilled water, pH 7.4. Others are commercially available.

0.4 % solution Trypan blue.

Bright-line hemacytometer.

Light microscope.

Fc blocking antibodies (CD16/CD32).

Antibodies applicable for flow cytometry.

Fixable Viability Dye (eBioscience, CA).

2 % Paraformaldehyde (PFA).

RPMI-1640 medium supplemented with 10 % fetal bovine serum, heat inactivated (FBS), and 1 % penicillin-streptomycin.

Phorbol 12-myristate 13-acetate (PMA).

Golgi Plug solution containing brefeldin A (BD Biosciences).

Humidified incubator with CO₂ tank.

Cytofix/Perm kit (BD Biosciences).

Flow cytometry acquisition instrument (e.g., 8-color updated BD FACS Calibur, BD Biosciences/Cytek).

FACS tubes with cell strainer caps.

Flow cytometry analysis software (e.g., FlowJo).

After euthanizing the mouse, remove all blood via cardiac puncture (see Note 1 ).

Open the abdominal and chest cavities and gently perfuse the aorta with PBS containing 2 % heparin to remove blood from the vessels.

Carefully remove any surrounding adipose tissue and dissect aorta (including branches within the arch), keeping the adventitia intact (see Note 2 ).

Place aorta in a FACS tube in 2 mL of PBS on ice.

Collect the spleen to prepare single-control samples for flow cytometer parameter settings (see step 4 in Subheading 3.3 and step 1 in Subheading 3.5 for details).

Place 35 μL of enzyme digestion solution in the FACS tube containing the aorta and 2 mL of PBS. Aortas can be cut into small pieces to improve digestion of the tissues.

Final enzyme digestion solution concentration (diluted in PBS) is:

Incubate for 45–60 min at 37 °C, and cover tubes with caps (see Note 3 ).

Use 70 μm cell strainer and syringe to release cells from digested aortic tissue returning cells to FACS tube.

Centrifuge at 368 rcf at 4 °C for 5 min, and then decant supernatant (see Notes 4 and 5 ).

Resuspend in 1 mL of PBS and place on ice.

For splenic tissue: Prepare single-cell suspension using a 70 μm cell strainer, remove red blood cells using lysis buffer, wash cells twice with PBS, then centrifuge, and decant as done with aortic samples. Resuspend in PBS.

Pipet or vortex the cell suspension to ensure that it is homogenous.

Count the number of aortic leukocytes using trypan blue to exclude nonviable cells.

Calculate the number of aortic leukocytes (see Note 6 ).

Centrifuge cells at 368 rcf, 4 °C for 5 min and then decant supernatant (see Note 5 ).

Following the same procedure, count the number of splenic leukocytes.

See Note 7 for an explanation of the appropriate controls to use.

If interested in intracellular staining, see Subheading 3.6 before proceeding with extracellular staining.

Perform staining on aortic cell suspension using a standard flow cytometry protocol. Briefly, incubate 1.0–1.5 × 10⁶ cells with 10 μg/ml of Fc blocking buffer (see Note 8 ) (0.5–1.0 × 10⁶ splenic cells should be used for each control sample).

Incubate for 10 min at room temperature to inhibit nonspecific binding of antibodies.

Add antibodies of interest at appropriate dilutions to 100 μL of diluted Fc blocking buffer and briefly vortex (see Notes 8 – 12 ).

If completing the viability staining, incorporate with the extracellular antibody staining here.

Incubate samples in the dark for 20 min at room temperature or 40 min at 4 °C.

After the antibody incubation, add 1 mL of PBS to wash.

Centrifuge at 368 rcf at 4 °C for 5 min and decant the supernatant (see Note 5 ).

Repeat steps 8 and 9.