Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

First, place a 15-milliliter conical tube into a rack. Place a sterile 250-micrometer metal mesh over the tube. Rinse the mesh with 1 milliliter of RPMI. Next, transfer the lymph nodes to the metal mesh, and use a pair of tweezers to grind them through the mesh. Apply 1 milliliter of RPMI on the mesh to flush the cells into the tube. Repeat this process of transferring and grinding the lymph nodes three times for each sample, and then, remove the mesh.

Centrifuge the tubes at 433 x g and at 4 degrees Celsius for 5 minutes. Discard the supernatant and resuspend the cells in approximately 5 milliliters of RPMI, then, fill the tubes with RPMI. Repeat this process from centrifuging the samples to filling the tube with RPMI one time.

Centrifuge the tubes again at 433 x g and at 4 degrees Celsius for 5 minutes. Discard the supernatant, and resuspend the cell pellet in 2 milliliters of RPMI. Transfer 2 milliliters of the cell suspension into 5-milliliter round-bottom tubes with cell strainer caps.

First, centrifuge the cell suspension from the PDLN samples at 433 x g and at 4 degrees Celsius for 5 minutes. Discard the supernatant. Stain the cells with surface antibodies, as outlined in the text protocol, and incubate the tubes on ice for 40 minutes.

Next, add 200 microliters of FACS buffer to each tube. Centrifuge at 433 x g and at 4 degrees Celsius for 5 minutes, and discard the supernatant. Repeat this process — adding the buffer, centrifuging, and discarding the supernatant one time. Resuspend the cell pellet in 500 microliters of permeabilization fixation buffer to fix and permeabilize the cells.

Transfer the tubes to a refrigerator at 4 degrees Celsius overnight. The next day, centrifuge the cell suspension from the PDLN samples at 433 x g and at 4 degrees Celsius for 5 minutes. Discard the supernatant, and resuspend the cell pellet in 500 microliters of permeabilization washing buffer. Centrifuge the cells again at 433 x g and at 4 degrees Celsius for 5 minutes, and discard the supernatant.

Stain the cells with intracellular antibodies, as outlined in the text protocol. Incubate the tubes on ice for 1 hour. After this, add 500 microliters of permeabilization washing buffer to each tube. Centrifuge at 433 x g and at 4 degrees Celsius for 5 minutes. Discard the supernatant, and resuspend the pellet in 500 microliters of permeabilization washing buffer.

Centrifuge once more at 433 x g in at 4 degrees Celsius for 5 minutes. Discard the supernatant, and resuspend the cell pellet in 300 microliters of FACS buffer. Then, analyze the cells on a flow cytometer as outlined in the text protocol.