Highly Multiplexed Single‐Cell Protein Analysis

Mondal, Manas; Liao, Renjie; Guo, Jia

doi:10.1002/chem.201705014

Cited by 19 publications

(18 citation statements)

References 88 publications

(187 reference statements)

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“…We next assessed the detection sensitivity of our approach by comparing it with direct and indirect immunofluorescence, which have similar sensitivity to the current multiplexed in situ protein profiling approaches 14 . Applying conventional immunofluorescence methods, we stained protein Ki67 in HeLa cells with Cy5 labeled primary antibodies ( Fig.…”

Section: Significantly Enhanced Detection Sensitivitymentioning

confidence: 99%

“…To enable multiplexed single-cell protein analysis, a number of methods have been explored recently, such as mass cytometry 9 , single cell barcode chips [10][11][12] , and DNAantibody barcoded arrays 13 . Nonetheless, as protein spatial complexity are masked in these approaches, they can not be applied to profile proteins in intact tissues in situ 14 . To address this issue, cyclical immunofluorescence [15][16][17][18][19][20][21][22][23] and mass cytometry imaging 24,25 have been developed.…”

Section: Introductionmentioning

confidence: 99%

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Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Liao

Mondal

Nazaroff

et al. 2019

Preprint

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The ability to comprehensively profile proteins in intact tissues in situ is crucial for our understanding of health and disease. However, the existing methods suffer from low sensitivity and limited sample throughput. To address these issues, here we present a highly sensitive and multiplexed in situ protein analysis approach using cleavable fluorescent tyramide and off-the-shelf antibodies. Compared with the current methods, this approach enhances the detection sensitivity and reduces the imaging time by 1-2 orders of magnitude, and can potentially detect hundreds of proteins in intact tissues at the optical resolution. Applying this approach, we studied protein expression heterogeneity in a population of genetically identical cells, and performed protein expression correlation analysis to identify coregulated proteins. We also profiled >6000 neurons in a human formalin-fixed paraffin-embedded (FFPE) hippocampus tissue. By partitioning these neurons into varied cell clusters based on their multiplexed protein expression profiles, we observed different subregions of the hippocampus consist of neurons from distinct clusters.

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Section: Significantly Enhanced Detection Sensitivitymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Liao

Mondal

Nazaroff

et al. 2019

Preprint

Self Cite

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“…These techniques preserve spatial information about RNA location, a useful feature when dealing with cells such as principal and ICs of the collecting duct with inter‐regulating phenotypes. Similarly, single cell proteomics and in situ single cell proteomics will prove to be informative.…”

Section: Discussionmentioning

confidence: 99%

Cellular plasticity: A mechanism for homeostasis in the kidney

et al. 2020

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Cellular plasticity is a topical subject with interest spanning a wide range of fields from developmental biology to regenerative medicine. Even the nomenclature is a subject of debate, and the underlying mechanisms are still under investigation. On top of injury repair, cell plasticity is a constant physiological process in adult organisms and tissues, in response to homeostatic challenges. In this review we discuss two examples of plasticity for the maintenance of homeostasis in the renal systemnamely the renin-producing juxtaglomerular cells (JG cells) and cortical collecting duct (CCD) cells. JG cells show plasticity through recruitment mechanisms, answering the demand for an increase in renin production. In the CCD, cells appear to have the ability to transdifferentiate between principal and intercalated cells to help maintain the highly regulated solute transport levels of that segment. These two cases highlight the complexity of plasticity processes and the role they can play in the kidney. K E Y W O R D Scollecting duct, JG cells, kidney, plasticity, renin 2 of 12 | ASSMUS et Al.

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“…With these further developments, C-FISH has the potential to enable single-cell in situ genomic or transcriptomic analysis. Finally, C-FISH can be combined with the single cell protein analysis technologies [ 36 , 37 ] to allow integrated DNA, RNA and protein analysis in individual cells in situ. This comprehensive molecular imaging platform will have wide applications in systems biology and precision medicine.…”

Section: Discussionmentioning

confidence: 99%

Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

2020

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The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

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Highly Multiplexed Single‐Cell Protein Analysis

Cited by 19 publications

References 88 publications

Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Highly sensitive in situ proteomics with cleavable fluorescent tyramide reveals human neuronal heterogeneity

Cellular plasticity: A mechanism for homeostasis in the kidney

Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

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