Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Regulatory T cells, or Tregs, are a specialized T cell subpopulation that exerts immunosuppressive effects to mediate immune system homeostasis. Tregs express the cell-surface markers CD4, CD25, and Neuropilin 1, Nrp1, and the nuclear transcription factors forkhead box P3, Foxp3, and Helios.

To isolate Tregs from mouse pancreatic draining lymph nodes, or PDLNs, place a metal mesh over a conical tube. Place the PDLNs on the mesh and grind the tissue to mechanically dissociate it. Add a suitable medium over the mesh, flushing the cells into the tube.

Centrifuge, removing dead cells and cellular debris. Resuspend the viable cells in the media. Pass the cells through a cell strainer cap fitted to a round-bottom tube to obtain a homogenous single-cell suspension, eliminating any remaining cell clumps.

Add fluorochrome-conjugated anti-CD4, anti-CD25, and anti-Nrp1 antibodies. The anti-CD4 and anti-CD25 antibodies bind to their respective cell-surface antigens on Tregs. Simultaneously, anti-Nrp1 binds to the Nrp1 receptors.

Treat the cells with a suitable permeabilization fixation buffer, fixing the cells and permeabilizing the cellular and nuclear membranes. Add fluorochrome-conjugated antibodies targeting Foxp3 and Helios.

The permeabilized membranes allow the antibodies to enter the cells and bind to their respective intranuclear targets. Analyze the cells using flow cytometry.

Tregs are characterized by the positive cell-surface expression of CD4, CD25, and Nrp1, along with the nuclear transcription factors Foxp3 and Helios.