Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is the leading cause of death from any infectious agent worldwide, with an estimated 10 million new cases in 2019. Drug development efforts for TB have classically relied on in vitro screening campaigns without consideration for Mtb's established intracellular lifestyle, which may not reflect true drug susceptibility in vivo. Here, we introduce two intracellular screening techniques based on the detection of different fluorescent markers to enumerate bacterial burden in THP-1 monocyte derived macrophages. These techniques are able to distinguish actively growing bacteria from killed bacteria by two distinct methodologies, with the use of cell wall intercalating dye DMN-Tre or an RFP expressing Mtb. This method may also be utilised in the screening of mutant Mtb libraries to evaluate the mutations’ effect on drug susceptibility and vice versa. As current high content platform technologies are able to perform a variety of functions, these techniques are broadly applicable to a multiplicity of intracellular screens. We further provide a comparison of infection techniques that may be used for drug screening (batch infection) and high content host–pathogen interaction analysis (2-day differentiation). The aim of this text is to provide the user with a solid and reproducible starting point to high content screening of intracellular Mtb, and to highlight adaptations to the protocol that may aid in future assay development.
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References
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Rankine-Wilson, L., Rens, C., Sahile, H.A., Av-Gay, Y. (2022). Mycobacterium tuberculosis Infection of THP-1 Cells: A Model for High Content Analysis of Intracellular Growth and Drug Susceptibility. In: Gal-Mor, O. (eds) Bacterial Virulence. Methods in Molecular Biology, vol 2427. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1971-1_7
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DOI: https://doi.org/10.1007/978-1-0716-1971-1_7
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1970-4
Online ISBN: 978-1-0716-1971-1
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