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With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH2) to construct NPs similar to unmodified PLGA NPs. PLGA-NH2NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [111In]In-PLGA-NH2showed higher specific activity than those labeled with [111In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureusinfection site in mice. In conclusion, PLGA-NH2NPs are able to retain 111In, independent of chelator presence. Furthermore, [111In]In-PLGA-NH2allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.