scholarly journals A Co-registration Pipeline for Multimodal MALDI and Confocal Imaging Analysis of Stem Cell Colonies

2020 ◽  
Vol 31 (4) ◽  
pp. 986-989
Author(s):  
Arina Nikitina ◽  
Danning Huang ◽  
Li Li ◽  
Nicholas Peterman ◽  
Sarah E. Cleavenger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Confocal Imaging ◽  
Imaging Analysis

scholarly journals SUPERMAN prevents stamen formation and promotes stem cell termination in the fourth whorl of the Arabidopsis flower

2017 ◽  
Author(s):  
Nathanaël Prunet ◽  
Weibing Yang ◽  
Pradeep Das ◽  
Elliot M. Meyerowitz ◽  
Thomas P. Jack
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Floral Organ ◽  
Genetic Networks ◽  
Confocal Imaging ◽  
Identity Change ◽  
Floral Organ Identity ◽  
Organ Identity

SummaryThe molecular and genetic networks underlying the determination of floral organ identity are well studied, but much less is known about how the flower is partitioned into four developmentally distinct whorls. The SUPERMAN gene is required for proper specification of the boundary between stamens in whorl 3 and carpels in whorl 4, as superman mutants exhibit supernumerary stamens but usually lack carpels. However, it has remained unclear whether extra stamens in superman mutants originate from an organ identity change in whorl 4 or the overproliferation of whorl 3. Using live confocal imaging, we show that the extra stamens in superman mutants arise from cells in whorl 4, which change their fate from female to male, while floral stem cells proliferate longer, allowing for the production of additional stamens.

Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ

1997 ◽  
Vol 272 (6) ◽  
pp. C1980-C1987 ◽  
Author(s):  
H. Ohata ◽  
Y. Ujike ◽  
K. Momose
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Cultured Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Imaging ◽  
Transient Increase ◽  
Similar Response ◽  
Imaging Analysis ◽  
Acetoxymethyl Ester

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

Confocal Imaging Analysis of Mitochondrial Trafficking of Individual Lipid Species in Live Cells

2021 ◽  
Author(s):  
Jun Zhang ◽  
Jia Nie ◽  
Haoran Sun ◽  
John-Paul Andersen ◽  
Yuguang Shi
Keyword(s):  
Confocal Imaging ◽  
Live Cells ◽  
Imaging Analysis ◽  
Lipid Species ◽  
Mitochondrial Trafficking

scholarly journals High-throughput Automated Single Cell Imaging Analysis Reveals Dynamics Of Glioblastoma Stem Cell Population During State Transition

2018 ◽  
Author(s):  
Anastasia P. Chumakova ◽  
Masahiro Hitomi ◽  
Erik P. Sulman ◽  
Justin D. Lathia
Keyword(s):  
Stem Cell ◽  
Protein Expression ◽  
Single Cell ◽  
High Throughput ◽  
Cell Imaging ◽  
State Transition ◽  
Imaging Analysis ◽  
Sox2 Expression ◽  
Single Cell Imaging ◽  
Stem Cell State

ABSTRACTCancer stem cells (CSCs) are a heterogeneous and dynamic population that stands at the top of tumor cellular hierarchy and is responsible for maintenance of the tumor microenvironment. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a High-throughput Automated Single Cell Imaging Analysis (HASCIA) approach for quantitative assessment of protein expression with single cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSC as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2-low cells, which expanded in fraction size during state transition. HASCIA also revealed that CSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with quantification of SOX2 expression by HASCIA defined a stable cut-off of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity and a presence of a sub-population of slowly proliferating SOX2-low CSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of CSCs and their progeny. It is a powerful and easy-to-use image analysis and statistical analysis tool available athttps://hascia.lerner.ccf.org.

Pathway and control of sucrose import into initiating cotton fibre cells

2000 ◽  
Vol 27 (9) ◽  
pp. 795 ◽  
Author(s):  
Yong-Ling Ruan ◽  
Danny J. Llewellyn ◽  
Robert T. Furbank
Keyword(s):  
Confocal Imaging ◽  
Assimilate Transport ◽  
Imaging Analysis ◽  
Sucrose Transport ◽  
Trichome Development ◽  
Fibre Cell ◽  
Key Enzyme ◽  
And Control ◽  
Symplastic Pathway ◽  
Unique Mechanism

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 Our aim is to unravel the mechanisms controlling fibre cell initiation from the epidermis of cotton (Gossypium hirsutum L.) ovules. We compared the development of fibres and trichomes in wild type cotton and a fibreless seed (fls) mutant, and determined the cellular pathway of sucrose transport into fibre initials on the day of anthesis. Although fibre initiation is inhibited in the fls mutant, leading to the fibreless phenotype, trichome development in other parts of the plant is normal. Confocal imaging analysis revealed that the fluorescent molecule, 5(6)-carboxyfluorescein, which is transported symplastically, moved readily from the integument phloem into initiating fibres. Plasmolysis studies showed that the fibre initials and adjacent non-initiating ovule epidermal cells have similar osmotic potential. Immunolocalisation analysis showed the absence of sucrose transporter proteins in the initiating fibre, but their abundance in the transfer cell precursors at the innermost integument. These results (i) demonstrate that fibre cell initiation is controlled by unique mechanism(s) that differ from that for normal trichome development; (ii) show a symplastic pathway of sucrose import into initiating fibres and strengthen the current opinion that sucrose synthase is likely to be the key enzyme mobilising sucrose into initiating fibres; and (iii) suggest that the initial protrusion of the fibre cells above the ovule surface is largely achieved by increased cell wall extensibility rather than higher turgor as is commonly thought.

scholarly journals Neutrophil Extracellular Traps (NETs) Contribute to the Formation of Microvascular Thrombosis in Immune Thrombotic Thrombocytopenic Purpura

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1020-1020
Author(s):  
Noritaka Yada ◽  
Jingrui Sui ◽  
Liang Zheng ◽  
X. Long Zheng
Keyword(s):  
Neutrophil Extracellular Traps ◽  
Dnase I ◽  
Confocal Imaging ◽  
Adamts13 Activity ◽  
Imaging Analysis ◽  
Thrombocytopenic Purpura ◽  
Pathogenetic Role ◽  
Extracellular Traps ◽  
Endothelial Surface

Abstract Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood. Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2). Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. Figure 1 Figure 1. Disclosures Zheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.

scholarly journals Differences in Stem Cell Marker and Osteopontin Expression in Primary and Recurrent Glioblastoma

2021 ◽  
Author(s):  
Bülent Polat ◽  
Gisela Wohlleben ◽  
Rebekka Kosmala ◽  
Dominik Lisowski ◽  
Frederick Mantel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Clinical Investigation ◽  
Recurrent Glioblastoma ◽  
Confocal Imaging ◽  
Stem Cell Marker ◽  
Stem Cell Markers ◽  
Recurrent Tumor ◽  
Isocitrate Dehydrogenase 1 ◽  
Recurrent Tumors

Abstract Background: Despite of a multimodal approach, recurrences can hardly be prevented in glioblastoma. This may be in part due to so called glioma stem cells. However, there is no established marker to identify these stem cells. Methods: Paired samples from glioma patients were analyzed by immunohistochemistry for expression of the following stem cell markers: CD133, Musashi, Nanog, Nestin, octamer-binding transcription factor 4 (Oct4), and sex determining region Y-box 2 (Sox2). In addition, the expression of osteopontin (OPN) was investigated. The relative number of positively stained cells was determined. By means of Kaplan-Meier analysis, a possible association with overall survival by marker expression was investigated. Results: Sixty tissue samples from 30 patients (17 male, 13 female) were available for analysis. For Nestin, Musashi and OPN a significant increase was seen. There was also an increase (not significant) for CD133 and Oct4. Patients with mutated Isocitrate Dehydrogenase-1/2 (IDH-1/2) status had a reduced expression for CD133 and Nestin in their recurrent tumors. Significant correlations were seen for CD133 and Nanog between OPN in the primary and recurrent tumor and between CD133 and Nestin in recurrent tumors. By confocal imaging we could demonstrate a co-expression of CD133 and Nestin within recurrent glioma cells. Patients with high CD133 expression had a worse prognosis (22.6 vs 41.1 months, p = 0.013). A similar trend was seen for elevated Nestin levels (24.9 vs 41.1 months, p = 0.08). Conclusions: Most of the evaluated markers showed an increased expression in their recurrent tumor. CD133 and Nestin were associated with survival and are candidate markers for further clinical investigation.

scholarly journals Bioimaging of Nucleolin Aptamer-Containing 5-(N-benzylcarboxyamide)-2′-deoxyuridine More Capable of Specific Binding to Targets in Cancer Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Kyue Yim Lee ◽  
Hyungu Kang ◽  
Sung Ho Ryu ◽  
Dong Soo Lee ◽  
Jung Hwan Lee ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Cancer Cells ◽  
Specific Binding ◽  
Confocal Imaging ◽  
Critical Parameters ◽  
Significant Activity ◽  
Imaging Analysis ◽  
Modified Nucleotides ◽  
Chemically Modified ◽  
Fluorescence Measurements

Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures.

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