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基于基因组的中等通量方案用于定量感染期间胞内分枝杆菌负荷和巨噬细胞存活。

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

机构信息

National Emerging Infectious Diseases Laboratories (NEIDL), Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston University, Boston, MA 02118, USA.

Department of Cancer Biology and Center for Cancer Systems Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

出版信息

STAR Protoc. 2022 Mar 15;3(2):101241. doi: 10.1016/j.xpro.2022.101241. eCollection 2022 Jun 17.

DOI:10.1016/j.xpro.2022.101241
PMID:35310069
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8931439/
Abstract

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).

摘要

在这里，我们提出了一种简化的方案，用于评估巨噬细胞中的胞内结核分枝杆菌（Mycobacterium tuberculosis，Mtb）载量。该方案描述了使用自动化显微镜同时评估巨噬细胞活力。此外，我们详细介绍了一种从多聚甲醛固定的巨噬细胞中快速、廉价且准确地提取分枝杆菌 DNA 的方法，用于定量分枝杆菌负荷。使用内参和巨噬细胞活力同时评估细菌负荷，可以精确量化干扰因素对 Mtb 和宿主细胞的影响，同时考虑技术伪影。如需了解本方案的使用和执行的完整详细信息，请参阅 Chatterjee 等人（2021 年）的研究。

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