Quantification of cytosolic DNA species by immunofluorescence microscopy and automated image analysis

Ai Sato, Norma Bloy, Claudia Galassi, Carlos Jiménez-Cortegana, Vanessa Klapp, Artur Aretz, Emma Guilbaud, Takahiro Yamazaki, Giulia Petroni, Lorenzo Galluzzi*, Aitziber Buqué

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma—but not nuclear or inner mitochondrial—membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.

Original languageEnglish
Title of host publicationRadiation Oncology and Radiotherapy Part A
EditorsAi Sato, Jeffrey Kraynak, Ariel E. Marciscano, Lorenzo Galluzzi
PublisherAcademic Press Inc.
Number of pages20
ISBN (Print)9780323899499
Publication statusPublished - Jan 2022
Externally publishedYes

Publication series

NameMethods in Cell Biology
ISSN (Print)0091-679X


  • Cancer immunotherapy
  • CGAS
  • FIJI
  • mtDNA
  • Type I interferon


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