Identifying Regulatory T Cell Subsets from Murine Pancreatic Draining Lymph Nodes

Overview

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.
4. Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
5. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
6. Resuspend the cell pellet in approximately 5 mL of RPMI. Fill the tubes with RPMI.
7. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
8. 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

1. Centrifuge the cell suspensions from PDLNs at 433 x g for 5 min at 4 °C. Discard the supernatant.
2. 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.
3. 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.
4. 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.
5. Keep the tubes in the fridge at 4 °C overnight.
   NOTE: A 1 h incubation also works.
6. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
7. 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.
8. 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.
9. Add 500 µL of PWB to each tube. Centrifuge the tubes at 433 x g for 5 min at 4 °C. Discard the supernatant.
10. 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.
11. Resuspend the cell pellet in 300 µL of FACS buffer.
12. Analyze the cells on a flow cytometer.
13. 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.
14. Export the FCS files of experiments and analyze them using flow cytometry analyzer software.

Materials

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