Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during in vitro infection

• Published in: STAR protocols Vol. 3; no. 2; p. 101241
• Main Authors: Yabaji, Shivraj M., Chatterjee, Sujoy, Waligursky, Emily, Gimelbrant, Alexander, Kramnik, Igor
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.06.2022; Elsevier
• Subjects: Cell-based Assays; Immunology; Macrophages - microbiology; Microbiology; Microscopy; Molecular Biology; Mycobacterium tuberculosis - genetics; Protocol; Immunology; Microbiology; Microscopy; Molecular Biology; Cell-based Assays
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101241

Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts. For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021). [Display omitted] •Rapid protocol for mycobacterial DNA isolation from fixed macrophages in 96-well format•Use of BCG spike and duplex qPCR for accurate quantification of Mtb loads•Simultaneous assessment of macrophage viability using automated microscopy in BSL2•Compatibility with advanced imaging and mycobacterial DNA-based applications Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts.