Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Single Cell Isolation from PDLN

1. Place a sterile 250 µm metal mesh over a 15 mL conical tube in a rack. Rinse the mesh with 1 mL of RPMI.
2. Transfer the lymph nodes to the metal mesh and grind them through the mesh using a pair of tweezers. Apply 1 mL of RPMI on the metal mesh to flush the cells into the tube. Repeat 3 more times and remove the mesh.
3. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
4. Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
5. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
6. Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
7. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
8. Resuspend the cell pellet in 2 mL of RPMI. Transfer 2 mL of the cell suspension to 5 mL round bottom tubes with cell strainer caps. Apply the cell suspension to the cell strainer caps.

2. Flow Cytometry

1. Centrifuge the cell suspensions from PDLNs at 433 x g for 5 min at 4 °C. Discard the supernatant.
2. Stain the cells with the following surface antibodies in 100 µL of flow cytometry staining buffer (FACS buffer): 0.75 µg/100 µL FITC-conjugated CD4 antibody, 0.60 µg/100 µL APC-H7-conjugated CD8 antibody, 0.30 µg/100 µL PE-conjugated CD25 antibody, 5 µL (1: 20) APC-conjugated Nrp1 antibody. Incubate the tubes on ice for 40 min.
3. Add 200 µL of FACS buffer to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant. Repeat one more time.
4. Fix and permeabilize the cells by resuspending the cell pellet in 500 µL of Permeabilization Fixation Buffer (PFB).
   NOTE: PFB is prepared by mixing 1 part of Fixation/Permeabilization Concentrate with 3 parts of Fixation/Permeabilization Diluent.
5. Keep the tubes in the fridge at 4 °C overnight.
   NOTE: A 1 h incubation also works.
6. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
7. Resuspend the cell pellet in 500 µL of Permeabilization Washing Buffer (PWB). Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
   NOTE: PWB is prepared by diluting Permeabilization Buffer (10x) to 1:10 in distilled water.
8. Stain the cells with the following intracellular antibodies in 100 µL of PWB: 0.30 µg/100 µL PE-Cy7-conjugated Foxp3 antibody, 4 µL (1: 25) PacificBlue-conjugated Helios antibody. Incubate the tubes on ice for 1 h.
9. Add 500 µL of PWB to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
10. Resuspend the cell pellet in 500 µL of PWB. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
11. Resuspend the cell pellet in 300 µL of FACS buffer.
12. Analyze the cells on a flow cytometer.
13. Gate the single cells based on Side Scatter-Area, Height, and Width, and Forward Scatter-Area, Height, and Width. Run one million events for analysis.
14. Export the FCS files of experiments and analyze them using flow cytometry analyzer software.