In this video, we describe a protocol for isolating single cells from murine pancreatic draining lymph nodes (PDLNs) and the characterization of regulatory T cells (Tregs) from the single-cell population using flow cytometry. Tregs are identified based on the positive expression of the cell-surface markers CD4, CD25, and Nrp1, along with the nuclear transcription factors Foxp3 and Helios.
Protocol
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 tub…
Disclosures
The authors have nothing to disclose.
Materials
RPMI-1640
Sigma-Aldrich
R0883-500ML
eBioscience Foxp3 / Transcription Factor-Staining Buffer Set
ThermoFisher
00-5523-00
Contains Fixation/Permeabilization Concentrate, Fixation/ Permeabilization Diluent and Permeabilization Buffer (10x)