Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method

We report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence, a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image

Jia-Ren Lin


Scholarcy highlights

  • Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, in the case of highly multiplexed methods such as mass cytometry able to correlate the levels of multiple signalling, differentiation and cell fate markers
  • With one channel used for image segmentation and registration, we find that 3–4 data channels is a typical limit for robust, high-throughput IF imaging in 96- and 384-well plates, when signals are relatively weak or fluorescent proteins are being imaged
  • In this paper we demonstrate the application of cyclic immunofluorescence to drug response at a single-cell level and show that multiplex single-cell measurement reveals new aspects of drug sensitivity and resistance; further refinement of the method will be reported at our web site at http://lincs.hms.harvard.edu/lin-NatCommun2015
  • The amount of information obtained at each cycle is higher than the channel number because each image is associated with multiple morphological features that can be automatically scored and analysed
  • CycIF data can be analysed using conventional imaging software or the algorithms recently developed for CyTOF data, but integrated analysis of multichannel intensity and morphometric data will require new tools
  • Future development of CycIF includes extending the number of cycles, validating more antibodies, applying the method to multi-cellular samples and tissues and improving analytical tools
  • The specific animal sources, catalogue numbers and dilutions for fluorophore-conjugated antibodies from Cell Signaling Technology are listed in Supplementary Table 1

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