Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Abstract

Source: Luo, Z., et al. Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry. J. Vis. Exp.  (2019)

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

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.
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)
eBioscience Flow Cytometry Staining Buffer	ThermoFisher	00-4222-26
CD4 Monoclonal Antibody (RM4-5), FITC, eBioscience	ThermoFisher	11-0042-85
CD25 Monoclonal Antibody (PC61.5), PE, eBioscience	ThermoFisher	12-0251-83
BD Pharmingen APC-H7 Rat antiMouse CD8a	BD Biosciences	560182
Mouse Neuropilin-1 APC-conjugated Antibody	R&D Systems	FAB5994A
FOXP3 Monoclonal Antibody (FJK-16s), PE-Cyanine7, eBioscience	ThermoFisher	25-5773-82
Pacific Blue anti-mouse/human Helios Antibody	BioLegend	137220
Falcon 5 mL Round-Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	CORNING	352235	A 35 µm nylon mesh is incorporated into the dual-position snap cap
Tube 15 mL, 120 mm x17 mm, PP	Sarstedt	62.554.002
Flow cytometry	BD
Flow cytometry analysis software	Inivai Technologies	Flowlogic