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

Publication ,  Journal Article
Cronan, MR; Matty, MA; Rosenberg, AF; Blanc, L; Pyle, CJ; Espenschied, ST; Rawls, JF; Dartois, V; Tobin, DM
Published in: Nat Methods
December 2018

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.

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Published In

Nat Methods

DOI

EISSN

1548-7105

Publication Date

December 2018

Volume

15

Issue

12

Start / End Page

1098 / 1107

Location

United States

Related Subject Headings

  • Zebrafish
  • Tuberculosis
  • Protein Kinase Inhibitors
  • Mycobacterium tuberculosis
  • Maleimides
  • Macrophages
  • Indoles
  • Imaging, Three-Dimensional
  • Image Processing, Computer-Assisted
  • Granuloma
 

Citation

APA
Chicago
ICMJE
MLA
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Cronan, M. R., Matty, M. A., Rosenberg, A. F., Blanc, L., Pyle, C. J., Espenschied, S. T., … Tobin, D. M. (2018). An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas. Nat Methods, 15(12), 1098–1107. https://doi.org/10.1038/s41592-018-0215-8
Cronan, Mark R., Molly A. Matty, Allison F. Rosenberg, Landry Blanc, Charlie J. Pyle, Scott T. Espenschied, John F. Rawls, Véronique Dartois, and David M. Tobin. “An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.Nat Methods 15, no. 12 (December 2018): 1098–1107. https://doi.org/10.1038/s41592-018-0215-8.
Cronan MR, Matty MA, Rosenberg AF, Blanc L, Pyle CJ, Espenschied ST, et al. An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas. Nat Methods. 2018 Dec;15(12):1098–107.
Cronan, Mark R., et al. “An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.Nat Methods, vol. 15, no. 12, Dec. 2018, pp. 1098–107. Pubmed, doi:10.1038/s41592-018-0215-8.
Cronan MR, Matty MA, Rosenberg AF, Blanc L, Pyle CJ, Espenschied ST, Rawls JF, Dartois V, Tobin DM. An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas. Nat Methods. 2018 Dec;15(12):1098–1107.

Published In

Nat Methods

DOI

EISSN

1548-7105

Publication Date

December 2018

Volume

15

Issue

12

Start / End Page

1098 / 1107

Location

United States

Related Subject Headings

  • Zebrafish
  • Tuberculosis
  • Protein Kinase Inhibitors
  • Mycobacterium tuberculosis
  • Maleimides
  • Macrophages
  • Indoles
  • Imaging, Three-Dimensional
  • Image Processing, Computer-Assisted
  • Granuloma