scholarly journals Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

2021 ◽  
Vol 22 (16) ◽  
pp. 8644
Author(s):  
Thai Pham ◽  
Christopher D. Nazaroff ◽  
Joshua Labaer ◽  
Jia Guo
Keyword(s):  
Single Cell Analysis ◽  
Expression Profiles ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Great Promise ◽  
Specific Cell ◽  
Protein Targets ◽  
Cell Clusters ◽  
Protein Imaging

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell–cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

scholarly journals Highly sensitive and multiplexed in situ protein profiling with cleavable fluorescent streptavidin

2019 ◽  
Author(s):  
Renjie Liao ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Jia Guo
Keyword(s):  
Signal Amplification ◽  
Individual Cell ◽  
Protein Detection ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

AbstractThe ability to perform highly sensitive and multiplexed in situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, here we develop an approach using cleavable biotin conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced by at least 10 fold, compared with the existing methods. After imaging, the fluorophores and the biotins unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be unambiguously detected in individual cell in situ.

scholarly journals Highly Sensitive and Multiplexed In-Situ Protein Profiling with Cleavable Fluorescent Streptavidin

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 852 ◽  
Author(s):  
Renjie Liao ◽  
Thai Pham ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
Joshua Labaer ◽  
...  
Keyword(s):  
Optical Resolution ◽  
Protein Detection ◽  
Protein Analysis ◽  
Protein Profiling ◽  
Detection Sensitivity ◽  
Layer By Layer ◽  
Protein Targets ◽  
Highly Sensitive ◽  
Wide Range

The ability to perform highly sensitive and multiplexed in-situ protein analysis is crucial to advance our understanding of normal physiology and disease pathogenesis. To achieve this goal, we here develop an approach using cleavable biotin-conjugated antibodies and cleavable fluorescent streptavidin (CFS). In this approach, protein targets are first recognized by the cleavable biotin-labeled antibodies. Subsequently, CFS is applied to stain the protein targets. Though layer-by-layer signal amplification using cleavable biotin-conjugated orthogonal antibodies and CSF, the protein detection sensitivity can be enhanced at least 10-fold, compared with the current in-situ proteomics methods. After imaging, the fluorophore and the biotin unbound to streptavidin are removed by chemical cleavage. The leftover streptavidin is blocked by biotin. Upon reiterative analysis cycles, a large number of different proteins with a wide range of expression levels can be profiled in individual cells at the optical resolution. Applying this approach, we have demonstrated that multiple proteins are unambiguously detected in the same set of cells, regardless of the protein analysis order. We have also shown that this method can be successfully applied to quantify proteins in formalin-fixed paraffin-embedded (FFPE) tissues.

scholarly journals Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2206
Author(s):  
Thai Pham ◽  
Renjie Liao ◽  
Joshua Labaer ◽  
Jia Guo
Keyword(s):  
High Performance ◽  
Single Cells ◽  
Protein Quantification ◽  
Cellular Systems ◽  
Protein Profiling ◽  
High Signal ◽  
Protein Targets ◽  
Chemical Reagents ◽  
Ffpe Tissues

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.

scholarly journals Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

2019 ◽  
Author(s):  
Arnav Moudgil ◽  
Michael N. Wilkinson ◽  
Xuhua Chen ◽  
June He ◽  
Alex J. Cammack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Single Cell ◽  
Binding Sites ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Cell Types ◽  
Specific Cell

AbstractIn situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

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

2019 ◽  
Author(s):  
Renjie Liao ◽  
Manas Mondal ◽  
Christopher D. Nazaroff ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Optical Resolution ◽  
Detection Sensitivity ◽  
Formalin Fixed Paraffin ◽  
Highly Sensitive ◽  
Formalin Fixed Paraffin Embedded ◽  
Low Sensitivity ◽  
Protein Expression Profiles

ABSTRACTThe 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 genetically identical cells, and performed expression correlation analysis to identify coregulated proteins. We also profiled >6000 neurons in human formalin-fixed paraffin-embedded (FFPE) hippocampus. By partitioning these neurons into varied cell clusters based on their protein expression profiles, we observed different subregions of the hippocampus consist of neurons from distinct clusters.

scholarly journals Highly Sensitive and Multiplexed Protein Imaging With Cleavable Fluorescent Tyramide Reveals Human Neuronal Heterogeneity

2021 ◽  
Vol 8 ◽  
Author(s):  
Renjie Liao ◽  
Manas Mondal ◽  
Christopher D. Nazaroff ◽  
Diego Mastroeni ◽  
Paul D. Coleman ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
Optical Resolution ◽  
Detection Sensitivity ◽  
Formalin Fixed Paraffin ◽  
Highly Sensitive ◽  
Formalin Fixed Paraffin Embedded ◽  
Low Sensitivity ◽  
Protein Expression Profiles

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 co-regulated proteins. We also profiled >6,000 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 sub-regions of the hippocampus consist of neurons from distinct clusters.

scholarly journals A systematic comparison of fibroblasts derived from postmortem human dura mater versus dermal epithelium for neurodegenerative disease modeling

2021 ◽  
Author(s):  
Andrea R. Argouarch ◽  
Celica G. Cosme ◽  
Kristle Garcia ◽  
Christian I. Corrales ◽  
Alissa L. Nana ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Cell Lines ◽  
Dura Mater ◽  
Disease Modeling ◽  
Chromosomal Abnormalities ◽  
Expression Profiles ◽  
Dermal Fibroblasts ◽  
High Rate ◽  
Great Promise ◽  
Specific Cell

Patient-derived cells hold great promise for precision medicine approaches in human health. Fibroblast cells have been a major source of human cells for reprogramming and differentiating into specific cell types for disease modeling. Such cells can be isolated at various stages during life (presymptomatic, symptomatic, and postmortem) and thus can potentially be used to model different phases of disease progression. In certain circumstances, however, tissues are not collected during life and only postmortem tissues are the only available source of fibroblasts. Fibroblasts cultured from postmortem human dura mater of individuals with neurodegenerative diseases have been suggested as a primary source of cells for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols requires further characterization. In this study, cells derived from dermal biopsies performed in living subjects were compared to cells derived from postmortem dura mater. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences between the dermis and dura mater-derived cell lines were found. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, exhibited slower growth rates, failed to express fibroblast protein markers, and exhibited significant differences in gene expression profiles. In addition, dura mater-derived cells were found to exhibit a high rate of chromosomal abnormalities, particularly in the loss of the Y chromosome. Our study highlights potential limitations of postmortem human dura mater-derived cells for disease modeling, argues for rigorous karyotyping prior to reprograming, and brings into question the identity of dura mater-derived cells as belonging to a fibroblast lineage.

scholarly journals Serum protein profiling reveals a specific upregulation of the immunomodulatory protein progranulin in COVID-19

2020 ◽  
Author(s):  
Marina Rieder ◽  
Luisa Wirth ◽  
Luisa Pollmeier ◽  
Maren Jeserich ◽  
Isabella Goller ◽  
...  
Keyword(s):  
Serum Protein ◽  
Interleukin 6 ◽  
Expression Profiles ◽  
Negative Control ◽  
Adverse Outcomes ◽  
Protein Profiling ◽  
Interferon Signaling ◽  
Virus Elimination ◽  
Immunomodulatory Protein ◽  
Viral Responses

Abstract Background Severe courses of COVID-19 are associated with elevated levels of interleukin 6. However, there is a growing body of evidence pointing to a broad and more complex disorder of pro-inflammatory and anti-viral responses with disturbed interferon signaling in COVID-19. Methods In this prospective single-center registry, we included SARS-CoV-2 positive patients and patients with similar symptoms and severity of disease but negative for SARS-CoV-2 admitted to the emergency department and compared their serum protein expression profiles. Results Interleukin-6 abundance was similar in SARS-CoV-2 positive patients (n = 24) compared to SARS-CoV-2 negative control (n = 61). In contrast, we observed a specific upregulation of the immunomodulatory protein progranulin (GRN). High GRN abundance was associated with adverse outcomes and increased expression of interleukin-6 in COVID-19. Conclusion The data from this registry reveals that GRN is specifically upregulated in SARS-CoV-2 positive patients while interleukin-6 may serve as marker for disease severity. The potential of GRN as a biomarker and a possible impact of increased GRN expression on interferon signaling, virus elimination, and virus-induced lung tissue damage in COVID-19 should be further explored.

scholarly journals Analysis of the transcriptome of bovine endometrial cells isolated by laser micro-dissection (1): specific signatures of stromal, glandular and luminal epithelial cells

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Wiruntita Chankeaw ◽  
Sandra Lignier ◽  
Christophe Richard ◽  
Theodoros Ntallaris ◽  
Mariam Raliou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Localization ◽  
Expression Profiles ◽  
Cell Types ◽  
Molecular Signature ◽  
Receptor Protein ◽  
Specific Gene ◽  
Specific Cell ◽  
Biological Processes ◽  
Endometrial Cells

Abstract Background A number of studies have examined mRNA expression profiles of bovine endometrium at estrus and around the peri-implantation period of pregnancy. However, to date, these studies have been performed on the whole endometrium which is a complex tissue. Consequently, the knowledge of cell-specific gene expression, when analysis performed with whole endometrium, is still weak and obviously limits the relevance of the results of gene expression studies. Thus, the aim of this study was to characterize specific transcriptome of the three main cell-types of the bovine endometrium at day-15 of the estrus cycle. Results In the RNA-Seq analysis, the number of expressed genes detected over 10 transcripts per million was 6622, 7814 and 8242 for LE, GE and ST respectively. ST expressed exclusively 1236 genes while only 551 transcripts were specific to the GE and 330 specific to LE. For ST, over-represented biological processes included many regulation processes and response to stimulus, cell communication and cell adhesion, extracellular matrix organization as well as developmental process. For GE, cilium organization, cilium movement, protein localization to cilium and microtubule-based process were the only four main biological processes enriched. For LE, over-represented biological processes were enzyme linked receptor protein signaling pathway, cell-substrate adhesion and circulatory system process. Conclusion The data show that each endometrial cell-type has a distinct molecular signature and provide a significantly improved overview on the biological process supported by specific cell-types. The most interesting result is that stromal cells express more genes than the two epithelial types and are associated with a greater number of pathways and ontology terms.

scholarly journals The Jekyll and Hyde of Cellular Senescence in Cancer

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 208
Author(s):  
Dilara Demirci ◽  
Bengisu Dayanc ◽  
Fatma Aybuke Mazi ◽  
Serif Senturk
Keyword(s):  
Cancer Cell ◽  
Cellular Senescence ◽  
Expression Profiles ◽  
Treatment Strategies ◽  
Gene Expression Profiles ◽  
Therapy Resistance ◽  
Disease Recurrence ◽  
Adverse Outcomes ◽  
Cell Senescence ◽  
Great Promise

Cellular senescence is a state of stable cell cycle arrest that can be triggered in response to various insults and is characterized by distinct morphological hallmarks, gene expression profiles, and the senescence-associated secretory phenotype (SASP). Importantly, cellular senescence is a key component of normal physiology with tumor suppressive functions. In the last few decades, novel cancer treatment strategies exploiting pro-senescence therapies have attracted considerable interest. Recent insight, however, suggests that therapy-induced senescence (TIS) elicits cell-autonomous and non-cell-autonomous implications that potentially entail detrimental consequences, reflecting the Jekyll and Hyde nature of cancer cell senescence. In essence, the undesirable manifestations that generally culminate in inflammation, cancer stemness, senescence reversal, therapy resistance, and disease recurrence are dictated by the persistent accumulation of senescent cells and the SASP. Thus, mitigating these pro-tumorigenic effects by eliminating these cells or inhibiting their SASP production holds great promise for developing innovative therapeutic strategies. In this review, we describe the fundamental aspects and dynamics of cancer cell senescence and summarize the comprehensive research on the adverse outcomes of TIS. Furthermore, we underline the rationale and motivation of emerging senotherapeutic modalities surrounding the removal of senescent cells and the SASP to help maximize the overall efficacy of cancer therapies.

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