Analysing single cell secretions by ‘shadow imaging’

Ashley Ambrose, Khodor Hazime, Daniel M Davis

Research output: Contribution to journalArticlepeer-review


Here, we describe a method, which we term ‘shadow imaging’, to analyse the secretions of individual cells at immune synapses, or other cell contacts. Following immune synapse formation and cellular activation on ligand-rich slides the position of each cell is recorded using a pulsed immunofluorescence stain against the proteins on the ligand-rich slide surface. The pulsed stain does not penetrate the synaptic cleft, resulting in an unlabelled region or ‘shadow’ beneath cells that is retained following cellular detachment. The secreted components, such as perforin, exosomes or other types of extracellular vesicles are retained on the slide and can be analysed on a single-cell basis using immunofluorescence. The ability to identify single cells secreting different combinations of particles, proteins and vesicles, enables us to better understand the heterogeneity in immune cell secretions, and can be used as a novel approach for phenotyping cell populations.
Original languageEnglish
JournalMethods in Molecular Biology
Publication statusAccepted/In press - 5 Dec 2022


  • Immune synapse
  • secretion
  • extracellular vesicles
  • cytotoxicity
  • single-cell analysis
  • bioimaging
  • immunofluorescence


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