Matching Items (3)

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Synthesis and Characterization of Mitochondria Targeting Fluorescent Probes

Description

A series of mitochondria targeting probes was synthesized for the purpose of exploring the feasibility of a mitochondria targeting fluorescent sensor. Of the probes, the probe with a two carbon

A series of mitochondria targeting probes was synthesized for the purpose of exploring the feasibility of a mitochondria targeting fluorescent sensor. Of the probes, the probe with a two carbon spacer showed the best co-localization from staining with the established MitoTracker Red® FM, indicating a potential development of the probe into mitochondria targeting sensor. However, cytotoxicity was observed for the probe with a six carbon spacer. Three additional mitochondria targeting fluorescent probes of longer spacer groups were synthesized, but the cytotoxicity was not observed to be as high as that of the probe with a two carbon spacer. The cytotoxicity was characterized to be that of caspase dependent cell death. To screen for a possible effect on apoptosis due to the mitochondrial probe, three fluorescent fusion proteins binding the anti-apoptotic proteins were designed and expressed. Each purified fusion protein was then incubated with the cytotoxic mitochondrial probe, and the mixture was isolated by running an affinity column. The fluorescence analysis of eluted fractions showed preliminary data of possible interaction between the protein and the mitochondrial probe.

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Date Created
  • 2014-12

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Highly Multiplexed Single Cell in situ Protein Analysis with Cleavable Fluorescent Probes

Description

Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on

Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.

An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.

This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus.

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Date Created
  • 2019

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Multiplexed single-cell spatial proteomics and transcriptomics

Description

Single-cell proteomics and transcriptomics analysis are crucial to gain insights of

healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide dee

Single-cell proteomics and transcriptomics analysis are crucial to gain insights of

healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment.

As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA and protein molecules in single cell in situ. Novel fluorescent probes have been designed and synthesized, which targets specifically either their nucleic acid counterpart or protein epitopes. These highly multiplexed imaging-based platforms have the potential to detect and quantify 100 different protein molecules and 1000 different nucleic acids in a single cell.

Using novel fluorescent probes, a large number of biomolecules have been detected and quantified in formalin-fixed paraffin-embedded (FFPE) brain tissue at single-cell resolution. By studying protein expression levels, neuronal heterogeneity has been revealed in distinct subregions of human hippocampus.

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Created

Date Created
  • 2018