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Summary: Macrophages are key innate immune cells involved in a broad spectrum of physiological and pathological processes. Macrophage depletion with clodronate-liposomes is commonly used to investigate in vivo functions of macrophages in mice. Here, we describe a protocol that combines the depletion of resident macrophages with the reconstitution of the mice with in vitro differentiated, lentivirus-transduced bone marrow-derived macrophages (BMDMs) in the context of an experimental sepsis model. This experimental strategy is easily adapted to other experimental designs.For complete details on the use and execution of this protocol, please refer to Du et al. (2020).