Cell Trafficking

Cell Tracker Orange (Invitrogen, Carlsbad, CA) and carboxyfluorescein succinimidyl ester (Invitrogen) were used to label OT-1 T cells. Cell Tracker Orange–labeled cells were injected IA, and carboxyfluorescein succinimidyl ester–labeled cells were injected IV into wild-type C57BL/6 mice with established B16-OVA tumors. Mice were sacrificed at 1 and 24 hours postinjection, and the liver, spleen, lymph nodes, lungs, and tumor were harvested. Tissues were manually disaggregated using a MediMachine (BD Biosciences) analyzed by flow cytometry or flash frozen in OCT compound, cryosectioned in 9 µm sections, stained with 4’,6-diamidino-2-phenylindole, mounted and examined by immunofluorescence microscopy for localization within tumor tissue.