Flow Cytometry and Confocal Microscopy for ROS Evaluation in Fish and Human Spermatozoa

ABSTRACT Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.

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Catechin isolated from faba beans (Vicia faba L.): insights from oxidative stress and hypoglycemic effect in yeast cells through confocal microscopy, flow cytometry, and in silico strategy

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2021.1945953 ◽

2021 ◽

pp. 1-11

Author(s):

Dhiraj Kumar Choudhary ◽

Navaneet Chaturvedi ◽

Amit Singh ◽

Abha Mishra

Keyword(s):

Oxidative Stress ◽

Flow Cytometry ◽

Confocal Microscopy ◽

Vicia Faba ◽

In Silico ◽

Yeast Cells ◽

Hypoglycemic Effect ◽

Vicia Faba L ◽

Faba Beans

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Characterization of Acanthamoeba–microsphere association by multiparameter flow cytometry and confocal microscopy

Cytometry Part A ◽

10.1002/cyto.a.20210 ◽

2006 ◽

Vol 69A (4) ◽

pp. 266-272 ◽

Cited By ~ 3

Author(s):

Edward A. G. Elloway ◽

Roger A. Bird ◽

Christopher J. Hewitt ◽

Steven L. Kelly ◽

Stephen N. Smith

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Multiparameter Flow Cytometry

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Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.10.1527379 ◽

1992 ◽

Vol 40 (10) ◽

pp. 1605-1612 ◽

Cited By ~ 68

Author(s):

G C Knowles ◽

C A McCulloch

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Actin Polymerization ◽

Direct Method ◽

Dnase I ◽

Fluorescence Probes ◽

Fluorescence Labeling ◽

Cell Type ◽

Fluorescence Spectrophotometry ◽

Actin Rearrangement

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.

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Imaging Cardiovascular and Lung Macrophages With the Positron Emission Tomography Sensor 64 Cu-Macrin in Mice, Rabbits, and Pigs

Circulation Cardiovascular Imaging ◽

10.1161/circimaging.120.010586 ◽

2020 ◽

Vol 13 (10) ◽

Cited By ~ 1

Author(s):

Matthias Nahrendorf ◽

Friedrich Felix Hoyer ◽

Anu E. Meerwaldt ◽

Mandy M.T. van Leent ◽

Max L. Senders ◽

...

Keyword(s):

Positron Emission Tomography ◽

Flow Cytometry ◽

Confocal Microscopy ◽

Pet Imaging ◽

Near Infrared ◽

Emission Tomography ◽

Imaging Biomarker ◽

Positron Emission ◽

Pulmonary Macrophages

Background: Macrophages, innate immune cells that reside in all organs, defend the host against infection and injury. In the heart and vasculature, inflammatory macrophages also enhance tissue damage and propel cardiovascular diseases. Methods: We here use in vivo positron emission tomography (PET) imaging, flow cytometry, and confocal microscopy to evaluate quantitative noninvasive assessment of cardiac, arterial, and pulmonary macrophages using the nanotracer 64 Cu-Macrin—a 20-nm spherical dextran nanoparticle assembled from nontoxic polyglucose. Results: PET imaging using 64 Cu-Macrin faithfully reported accumulation of macrophages in the heart and lung of mice with myocardial infarction, sepsis, or pneumonia. Flow cytometry and confocal microscopy detected the near-infrared fluorescent version of the nanoparticle ( VT680 Macrin) primarily in tissue macrophages. In 5-day-old mice, 64 Cu-Macrin PET imaging quantified physiologically more numerous cardiac macrophages. Upon intravenous administration of 64 Cu-Macrin in rabbits and pigs, we detected heightened macrophage numbers in the infarcted myocardium, inflamed lung regions, and atherosclerotic plaques using a clinical PET/magnetic resonance imaging scanner. Toxicity studies in rats and human dosimetry estimates suggest that 64 Cu-Macrin is safe for use in humans. Conclusions: Taken together, these results indicate 64 Cu-Macrin could serve as a facile PET nanotracer to survey spatiotemporal macrophage dynamics during various physiological and pathological conditions. 64 Cu-Macrin PET imaging could stage inflammatory cardiovascular disease activity, assist disease management, and serve as an imaging biomarker for emerging macrophage-targeted therapeutics.

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Evaluation of Alternative Methods to Assess the Biological Properties of Propolis on Metabolic Activity and Biofilm Formation in Streptococcus mutans

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2019/1524195 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8

Author(s):

Jorge Jesús Veloz ◽

Marysol Alvear ◽

Luis A. Salazar

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Streptococcus Mutans ◽

Metabolic Activity ◽

Crystal Violet ◽

Biofilm Formation ◽

Biological Activities ◽

Biological Properties ◽

Alternative Methods ◽

Flow Cytometry Analysis

Several biological activities have been reported for the Chilean propolis, among their antimicrobial and antibiofilm properties, due to its high polyphenol content. In this study, we evaluate alternative methods to assess the effect of Chilean propolis on biofilm formation and metabolic activity of Streptococcus mutans (S. mutans), a major cariogenic agent in oral cavity. Biofilm formation was studied by using crystal violet and by confocal microscopy. The metabolic activity of biofilm was evaluated by MTT and by flow cytometry analysis. The results show that propolis reduces biofilm formation and biofilm metabolic activity in S. mutans. When the variability of the methods to measure biofilm formation was compared, the coefficient of variation (CV) fluctuated between 12.8 and 23.1% when using crystal violet methodology. On the other hand, the CV ranged between 2.2 and 3.3% with confocal microscopy analysis. The CV for biofilm’s metabolic activity measured by MTT methodology ranged between 5.0 and 11.6%, in comparison with 1.9 to 3.2% when flow cytometry analysis was used. Besides, it is possible to conclude that the methods based on colored compounds presented lower precision to study the effect of propolis on biofilm properties. Therefore, we recommend the use of flow cytometry and confocal microscopy in S. mutans biofilm analysis.

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