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NCL Method ITA-3

By Timothy Potter, Barry Neun, Anna Ilinskaya, Marina Dobrovolskaia

Mouse Granulocyte-Macrophage Colony-Forming Unit Assay

Listed in Datasets | publication by group NCL Protocols

Version 2.0b - published on 24 Jul 2020 doi:10.17917/YTHD-KF51 - cite this

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Description

Myelosuppression, a condition where bone marrow activity is reduced, is a common dose limiting toxicity of cytotoxic oncology drugs. Nanoparticles have the potential to distribute to the bone marrow and/or release drug that is delivered to bone marrow. Therefore, understanding the potential toxicity of nanoparticles and the drugs they carry is an important step in preclinical safety evaluation.  

Hematopoietic stem cells of the bone marrow (BM) proliferate and differentiate to form discrete cell clusters or colonies. This document describes a protocol for quantitative analysis of granulocyte-macrophage (GM) colony-forming units (CFU), employing murine BM. The protocol can be used for both in vitro and ex vivo analyses. The in vitro protocol involves isolation of bone marrow cells from healthy animals, followed by treatment in vitro with nanoparticle formulations. For ex vivo analysis, the bone marrow is isolated from animals injected with the nanoparticle formulation. The in vitro protocol does not account for nanoparticle biodistribution; however, in cases where dose information is not yet available and/or the nanoparticle formulation is still in the early phases of development, the in vitro protocol allows for a rapid screening of potentially toxic nanoparticle formulations. Furthermore, the in vitro protocol can also provide a quick estimation of the myelosuppressive potential of a cytotoxic oncology drug bound to a nanoparticle surface in comparison to a traditional formulation of the same cytotoxic oncology drug (see Figure 1).

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