title: Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.
abstract: Single-cell analysis reveals aspects of cellular physiology not evident from population-based studies, particularly in the case of highly multiplexed methods such as mass cytometry (CyTOF) able to correlate the levels of multiple signalling, differentiation and cell fate markers. Immunofluorescence (IF) microscopy adds information on cell morphology and the microenvironment that are not obtained using flow-based techniques, but the multiplicity of conventional IF is limited. This has motivated development of imaging methods that require specialized instrumentation, exotic reagents or proprietary protocols that are difficult to reproduce in most laboratories. Here we report a public-domain method for achieving high multiplicity single-cell IF using cyclic immunofluorescence (CycIF), a simple and versatile procedure in which four-colour staining alternates with chemical inactivation of fluorophores to progressively build a multichannel image. Because CycIF uses standard reagents and instrumentation and is no more expensive than conventional IF, it is suitable for high-throughput assays and screening applications.
authors.affiliation: HMS LINCS Center &Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA.
authors.forename: Jia-Ren
authors.initials: JR
authors.lastname: Lin
authors.affiliation: Department of Systems Biology Harvard Medical School 200 Longwood Avenue, Boston, Massachusetts 02115 USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Fallahi-Sichani
authors.affiliation: HMS LINCS Center &Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA.|Department of Systems Biology Harvard Medical School 200 Longwood Avenue, Boston, Massachusetts 02115 USA.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Sorger
mesh_terms: D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D059010:Single-Cell Analysis / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes