An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas

Mark R. Cronan, Molly A. Matty, Allison F. Rosenberg, Landry Blanc, Charlie J. Pyle, Scott T. Espenschied, John F. Rawls, Véronique Dartois, David M. Tobin

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.

Original languageEnglish (US)
Pages (from-to)1098-1107
Number of pages10
JournalNature Methods
Issue number12
StatePublished - Dec 1 2018

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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