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
- Place a sterile 250 µm metal mesh over a 15 mL conical tube in a rack. Rinse the mesh with 1 mL of RPMI.
- 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.
- Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
- Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
- Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
- Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
- Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
- 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
- Centrifuge the cell suspensions from PDLNs at 433 x g for 5 min at 4 °C. Discard the supernatant.
- 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.
- 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.
- 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.
- Keep the tubes in the fridge at 4 °C overnight.
NOTE: A 1 h incubation also works.
- Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
- 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.
- 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.
- Add 500 µL of PWB to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
- 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.
- Resuspend the cell pellet in 300 µL of FACS buffer.
- Analyze the cells on a flow cytometer.
- 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.
- Export the FCS files of experiments and analyze them using flow cytometry analyzer software.
