Quantitative Monitoring Spatiotemporal Activation of Ras and PKD1 Using Confocal Fluorescent Microscopy

Alzheimer’s disease (AD) and sporadic Creutzfeldt–Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins—amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and “compound” plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer’s association guidelines, and prion protein subtype with codon 129 methionine–valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.

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pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.6.h1937 ◽

1998 ◽

Vol 275 (6) ◽

pp. H1937-H1947 ◽

Cited By ~ 2

Author(s):

Barbara J. Muller-Borer ◽

Hua Yang ◽

Sayed A. M. Marzouk ◽

John J. Lemasters ◽

Wayne E. Cascio

Keyword(s):

Free Acid ◽

Fluorescent Microscopy ◽

Ventricular Myocardium ◽

Dual Emission ◽

Arterial Perfusion ◽

Butanedione Monoxime ◽

Different Depths ◽

Endocardial Surface ◽

Confocal Fluorescent Microscopy

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 μm were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

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P517Automated segmentation of gap junctions using confocal fluorescent microscopy

Cardiovascular Research ◽

10.1093/cvr/cvu091.189 ◽

2014 ◽

Vol 103 (suppl 1) ◽

pp. S95.1-S95

Author(s):

T Szepe ◽

M Kovacs ◽

GY Seprenyi ◽

A Vegh

Keyword(s):

Gap Junctions ◽

Fluorescent Microscopy ◽

Confocal Fluorescent Microscopy

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Cisplatin Fails To Stimulate Production Of Reactive Oxygen Species and Release Of Neutrophil Extracellular Traps By Human Neutrophils In Vitro

Blood ◽

10.1182/blood.v122.21.4714.4714 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4714-4714

Author(s):

Leonardo Pasalic ◽

Campbell Heather ◽

Shane Thomas ◽

Vivien M Chen

Keyword(s):

Reactive Oxygen Species ◽

Fluorescent Microscopy ◽

Neutrophil Extracellular Traps ◽

Vehicle Control ◽

Human Neutrophils ◽

Oxygen Species ◽

Extracellular Traps ◽

Spontaneous Production ◽

Confocal Fluorescent Microscopy

Background Cisplatin is a commonly used antineoplastic agent for treatment of a broad range of cancers. Cisplatin-based treatment has been associated with a significant risk of venous thromboembolism. The mechanisms through which cisplatin contributes to a prothrombotic state remain unclear. Neutrophil extracellular traps (NETs) consist of web-like DNA–histone core decorated with granule proteins and are released from activated neutrophils in a process dependent on reactive oxygen species (ROS), in particular hypochlorous acid (HOCl). Recently, NETs have been shown to play an important role in initiation and propagation of venous thrombus in a number of animal models of deep vein thrombosis. The aim of this study was to investigate whether NETs may provide a potential link between cisplatin and venous thromboembolism. Methods and Results To assess the effect of cisplatin on release of NETs by ex vivo human neutrophils isolated by positive immunomagnetic selection we visualised NETs release by confocal fluorescent microscopy and performed fluorimetric quantification of cell-free DNA (CFDNA) using either SYTOX Green nucleic acid stain (10 µM) or an ultrasensitive fluorescent assay Picogreen Quant IT (Invitrogen). In contrast to stimulation with phorbol 12-myristate 13-acetate (PMA) (25 nM),which resulted in 22 ng/104neutrophils of detectable CFDNA, neither of these two assays could detect any significant release of CFDNA by human neutrophils exposed to cisplatin (15 µM) for 2 or 4 hours above baseline similar with vehicle control. Furthermore, confocal fluorescent microscopy imaging of neutrophils stained with non-cell permeable DNA dye SYTOX Red (Invitrogen) demonstrated no difference in NET formation between control and cisplatin treated human neutrophils. Thus we could not demonstrate that NETS are produced in response to cisplatin treatment. In view of consistent reports that NET formation is ROS dependent we decided to investigate whether cisplatin exposure leads to production of ROS by human neutrophils. Few published studies into the effects of cisplatin on the production of ROS by human neutrophils in vitro offer conflicting results. We used flow cytometry and fluorescent probe hydroethidine (HE) for detection of intercellular superoxide anion radical in HL60 granulocytic cells in the presence of cisplatin (up to 50 µM). Differentiation down the granulocytic lineage after stimulation with ATRA was confirmed by light microscopy and by flow cytometry. Capacity of differentiated HL60 cells to generate NET formation after PMA stimulation was confirmed by fluorescence microscopy. Cisplatin failed to augment the spontaneous production of ROS by ATRA differentiated HL60 cells. The number of viable ethidium-high cells in cisplatin treated group did not differ from the vehicle control indicating no detectable production of ROS in response to cisplatin. In contrast, positive control treatment with PMA (25 nM) and menadione (40 µM) resulted in 4- and 20-fold increase in viable ethidium-high population respectively. ROS generation by human neutrophils was measured by a colorimetric assay for chlorination of extracellular taurine to determine if exposure to cisplatin results in the production of HOCl by human neutrophils in vitro. Treatment of resting neutrophils with cisplatin (15 µM) for 30 min or 120 min was not associated with an increase in the spontaneous production of HOCl above the baseline. Furthermore, the PMA (25 nM)-activated generation of HOCl production was not increased by pre-treating neutrophils with cisplatin indicating that there was no potentiation of ROS by pre-treatment with cisplatin. Discussion and Conclusion Our results suggest that cisplatin fails to induce release of NETs or HOCl from human neutrophils in vitro. These negative findings seem to be at odds with the well described pro-oxidative actions of cisplatin. One possible explanation centres on reported findings that the pro-oxidative effects of cisplatin are dependent on the mitochondrial generation of ROS whilst the mitochondria-generated ROS appear not to be instrumental to NET formation. Therefore, we postulate that cisplatin may not be able to induce NET formation by human neutrophils, which are known to contain few mitochondria, due to a sub-threshold ROS signal. Therefore it appears that cisplatin-associated increased risk of venous thrombosis is unlikely to be mediated through NETs. Disclosures: No relevant conflicts of interest to declare.

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Examination of Blood-Brain Barrier Transferrin Receptor by Confocal Fluorescent Microscopy of Unfixed Isolated Rat Brain Capillaries

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1998.70020883.x ◽

2002 ◽

Vol 70 (2) ◽

pp. 883-886 ◽

Cited By ~ 47

Author(s):

Jörg Huwyler ◽

William M. Pardridge

Keyword(s):

Rat Brain ◽

Blood Brain Barrier ◽

Transferrin Receptor ◽

Fluorescent Microscopy ◽

Brain Barrier ◽

Brain Capillaries ◽

Blood Brain ◽

Confocal Fluorescent Microscopy ◽

Isolated Rat

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2187 THE USE OF FIBEROPTIC CONFOCAL FLUORESCENT MICROSCOPY IN A MURINE MODEL OF MICROTESE

The Journal of Urology ◽

10.1016/j.juro.2011.02.2425 ◽

2011 ◽

Vol 185 (4S) ◽

Author(s):

Ryan Smith ◽

Parviz Kavoussi ◽

Gregory Lowe ◽

Raymond Costabile ◽

William Steers ◽

...

Keyword(s):

Murine Model ◽

Fluorescent Microscopy ◽

Confocal Fluorescent Microscopy

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TMIC-50. 3D QUANTIFICATION OF DYNAMIC CYTOKINE GRADIENTS PRODUCED BY PRIMARY TUMOR MODELS

Neuro-Oncology ◽

10.1093/neuonc/noz175.1084 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi258-vi259

Author(s):

Eric McGhee ◽

Juan Uruena ◽

Alex McGhee ◽

Duane Mitchell ◽

Catherine Flores ◽

...

Keyword(s):

Immune Cell ◽

Fluorescent Microscopy ◽

Chemotherapeutic Agents ◽

Integrated System ◽

Fluorescence Measurement ◽

Tumor Models ◽

Multiple Tumor ◽

Wide Range ◽

Confocal Fluorescent Microscopy

Abstract BACKGROUND & SIGNIFICANCE The immune response is a coordinated effort directed by cytokine gradients and concentrations. High sensitivity and spatial resolution are necessary to resolve cytokine gradients in 3-dimensions. This system uses in situ confocal fluorescent microscopy with printed bead-based immunoassays. The combination of 3D printing of the beads and biofabrication of patient derived tumors allows for direct imaging, quantification, and movies of tumor cytokine secretion in response to challenges from immune cells, tumor associated fibroblasts, and chemotherapeutic agents. HYPOTHESIS: Cytokine dynamics can be measured in real-time by balancing concentrations of detection antibodies in solution with stationary immunoassay beads with measured capture and release rates. Favorable balancing of assay kinetics can facilitate measurements of concentrations of cytokines between 50 pg/mL to over 2,000 pg/mL. METHODS In situ confocal fluorescent microscopy identifies position, fluorescence, and type of bead relative to the in vitro tumor. Cytokines including IL2, IL6, IL8, IL11, and IFNg and growth factors such as VEGF and TGFb have been measured for a wide range of solid tumors (brain, sarcoma, and epithelial) under different stresses. RESULTS Dynamic gradients of IL8 were found across multiple tumor models showing local concentrations in excess of 2,000 pg/mL. Production rates were estimated to be over 1 protein per second for each cell, and inclusion of cancer associated support cells showed 10x increases in some cytokines. CD4+ cell production of IL2 was confirmed and quantified and showed strong sensitivity to tumor activity and antigen presentation. Multiplexed beads of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 allow for simultaneous measurement of multiple chemokines. Uncertainties associated with fluorescence measurement and quantification of concentrations will be discussed. CONCLUSIONS This integrated system of 3D assay printing, biofabrication of tumors and immune cell constructs, and in situ confocal microscopy provides the first direct measurements of 3D cytokine gradients in response to tumor stress.

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