Fluorescent Cell Labeling Techniques

The fluorescent dyes 5(6 (link))-CFDA, SE (CFSE), (5-(and−6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine) (CMTMR, CellTracker orange) and Chloromethyl-coumarin (CMAC, CellTracker blue) were purchased from ThermoFisher (Basel, Switzerland) and the cell proliferation dye eFluor 670 was from eBioscience (San Diego, CA). mAb against PNAd (MECA-79) was from nanotools (Freiburg, Germany) and coupled to AlexaFluor-633 using a Protein Labeling Kit from Molecular Probes (Basel, Switzerland). Fc receptor blocking mAb (clone 2.4G2, 4.5 mg/mL) in FACS buffer (D-PBS supplemented with 1% milk powder and 0.1% NaN3) was produced in-house. T cells were fixed with 1% paraformaldehyde (PFA; Electron Microscopy Sciences, Lucerne, Switzerland) diluted in D-PBS. D-PBS supplemented with 10 mM EDTA was used for homogenizing LNs for flow cytometry experiments.