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.
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
2. Flow Cytometry
The authors have nothing to disclose.
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 |