Study of Cell Metabolic Activities Using Fluorescence Microscopy

2003 ◽  
Author(s):  
Dawn M. Sabados ◽  
Lisa X. Xu ◽  
Jay P. Gore
Keyword(s):  
Endothelial Cells ◽  
Laser Excitation ◽  
Cultured Cells ◽  
Tissue Level ◽  
Excitation Wavelength ◽  
Level Laser ◽  
Comparison Results ◽  
Normal Rat ◽  
Breast Cells ◽  
Metabolic Activities

In the present study, cultured normal rat microvessel endothelial cells are first used to explore the feasibility of studying the cell metabolic activities by detecting autofluorescence intensities. A mercury lamp is used to induce autofluorescence emissions in cells. Under an excitation wavelength of 360nm, the emissions peak at 470 nm corresponds with the autofluorescence signature of NAD(P)H, the concentration of which has been linked to the metabolic rate of the cell. A relation between the emission intensity and the NAD(P)H concentration is established. Based upon the results and observations of the endothelial cells, a protocol is developed for studying the autofluorescence of other cultured cells. It has been long known that cancer cells divide much more rapidly than normal cells depending on the degree of the tumor malignancy. The differences in metabolic rates of normal and cancerous breast cells are explored as they relate to autofluorescence emissions at different wavelengths and intensities. The comparison results can be used as a guideline to determine whether cells are cancerous. At a tissue level, laser excitation is probably needed to obtain measurable autofluorescence signals. The potential of autofluorescence measurement as a breast cancer diagnostic tool is discussed.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Vol 116 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Erol C. Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Author(s):  
Erol Can Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

Intense Photoluminescence and Photoluminescence Enhancement of Silicon Nanocrystals by Ultraviolet Irradiation

2007 ◽  
Vol 31 ◽  
pp. 74-76 ◽  
Author(s):  
P.T. Huy ◽  
P.H. Duong
Keyword(s):  
Silicon Nanocrystals ◽  
Room Temperature ◽  
Laser Excitation ◽  
Uv Light ◽  
Irradiation Time ◽  
Excitation Wavelength ◽  
Nonradiative Recombination ◽  
Emission Yield ◽  
Recombination Centers ◽  
Pl Intensity

Photoluminescence (PL) from silicon nanocrystals deposited on top of silica-glass template and from silicon nanocrystals in nc_Si/SiO2 multilayer films were studied as a function of ultraviolet (UV) laser irradiation time in vacuum. Both the films exhibit intense visible PL at room temperature under laser excitation. It was found that upon prolong irradiation time using a He-Cd laser (325 nm) the PL intensity of the films was spectacularly enhanced. The process is reversible and does not happen with excitation wavelength longer than 400 nm. Upon introducing air into the measurement chamber, a rapid decrease of the PL intensity was recorded. This observation suggests that the UV light may lead to modification of nonradiative recombination centers in the films and thus improves the emission yield of silicon nanocrystals.

Raman spectra of twisted bilayer graphene close to the magic angle

2D Materials ◽  
2022 ◽  
Author(s):  
Tiago Campolina Barbosa ◽  
Andreij C. Gadelha ◽  
Douglas A. A. Ohlberg ◽  
Kenji Watanabe ◽  
Takashi Taniguchi ◽  
...  
Keyword(s):  
Raman Spectra ◽  
Laser Excitation ◽  
Bilayer Graphene ◽  
Geometrical Model ◽  
Excitation Wavelength ◽  
Magic Angle ◽  
Phonon Interaction ◽  
Electron Phonon Interaction ◽  
Excitation Laser ◽  
Twisted Bilayer Graphene

Abstract In this work, we study the Raman spectra of twisted bilayer graphene samples as a function of their twist-angles (θ), ranging from 0.03º to 3.40º, where local θ are determined by analysis of their associated moiré superlattices, as imaged by scanning microwave impedance microscopy. Three standard excitation laser lines are used (457, 532, and 633 nm wavelengths), and the main Raman active graphene bands (G and 2D) are considered. Our results reveal that electron-phonon interaction influences the G band's linewidth close to the magic angle regardless of laser excitation wavelength. Also, the 2D band lineshape in the θ < 1º regime is dictated by crystal lattice and depends on both the Bernal (AB and BA) stacking bilayer graphene and strain soliton regions (SP) [1]. We propose a geometrical model to explain the 2D lineshape variations, and from it, we estimate the SP width when moving towards the magic angle.

INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"

1987 ◽  
Author(s):  
N Sakuragawa ◽  
S Saitoh ◽  
K Takahashi
Keyword(s):  
Endothelial Cells ◽  
Room Temperature ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Binding Activity ◽  
Endothelial Cell Culture ◽  
Antithrombin Activity ◽  
Thrombin Activity ◽  
Cultured Endothelial Cells ◽  
At Iii

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.

Molecular adaptation of vascular endothelial cells to oxidative stress

1993 ◽  
Vol 264 (3) ◽  
pp. C715-C722 ◽  
Author(s):  
D. Lu ◽  
N. Maulik ◽  
I. I. Moraru ◽  
D. L. Kreutzer ◽  
D. K. Das
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Cultured Cells ◽  
Vascular Endothelial Cells ◽  
Molecular Adaptation ◽  
Vascular Endothelial ◽  
Trypan Blue Exclusion ◽  
Two Dimensional Gel Electrophoresis ◽  
H2o2 Treatment ◽  
Trypan Blue Exclusion Test

Cellular organisms respond at the cellular and molecular level when confronted with sudden changes in environment, and molecular adaptation represents the ability of the cells to acclimate themselves to their new environment. In this study we examined the response of bovine vascular endothelial cells (VEC) to the oxidative stress by exposing the cultured cells to two different concentrations of H2O2, 0.04 or 0.08 mM, for 18-24 h. H2O2-exposed VEC displayed good viability (85-90% for 0.04 mM H2O2; 75-80% for 0.08 mM H2O2) and exhibited normal morphology. H2O2 treatment of the VEC was associated with the expression of a number of new proteins, as demonstrated by two-dimensional gel electrophoresis of total cell lysate. Cells exposed to 0.04 mM H2O2 expressed 25 new proteins, whereas 19 newly expressed proteins were detected when the cells were exposed to 0.08 mM H2O2. Western blot analysis of H2O2-treated VEC using specific antibodies to heat-shock proteins (HSP) identified one of these proteins as a member of the HSP 70 family. In addition, H2O2 induced an increase in antioxidative enzyme activities in the VEC, including superoxide dismutase, catalase, and glutathione peroxidase. Moreover, these changes were a truly adaptive phenomenon because challenging the VEC with brief exposure to toxic levels of H2O2 (1 mM for 30 min) showed increased viability (by Trypan blue exclusion test) and decreased injury (by lactate dehydrogenase supernatant-to-cellular ratio determination) in adapted cells (preexposed to 0.04 or 0.08 mM H2O2) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of Antithrombin III in Fresh and Cultured Endothelial Cells from Human Umbilical Cord

1979 ◽  
Author(s):  
Vivian Chan ◽  
T.K. Chan
Keyword(s):  
Endothelial Cells ◽  
Umbilical Cord ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Active Role ◽  
Human Umbilical Cord ◽  
Thrombosis Research ◽  
Natural Inhibitor ◽  
Immunofluorescent Technique ◽  
Radio Immunoassay

We have shown by immunofluorescent technique that the distribution of antithrombin III (ATIII) in human tissues was concentrated around the microvasculature of the lungs and kidneys, as well as veins and small arteries of other organs (liver and spleen). It would seem that ATIII is stored and/or synthesized in the endothelial cells similar to Factor VIII-RAG and Plasminogen Activator. Endothelial cells were isolated from human umbilical cord by collagenase and cultured according to Chemethod described by Shearn etal (1977). In freshly isolated endothelial cells, ATIII could be demonstrated by indirect immunof1uorescent technique and radio immunoassay confirmed the presence of 14.8 ng per 106 cells. After 7 days’ culture, the supernatant from 106 cells contained about 15 ng and the cultured cells (106) contained 16.9 ng ATIII. The presence of ATIII in cultured cells was also confirmed by the positive immunofluorescence. Hence the endothelial cells play an active role in preventing thrombosis by the synthesis and liberation of ATIII, the major natural inhibitor of the intrinsic pathway of Coagulation.Reference: Shearn S.A., Peake I.R., Ciddings J.C., Humphrys J. and Bloom A.L. Thrombosis Research, 11, 43, 1977.

Oxidative stress in primary culture hepatocytes isolated from partially hepatectomized rats

2007 ◽  
Vol 85 (10) ◽  
pp. 1047-1051 ◽  
Author(s):  
Daniel Francés ◽  
M. Teresa Ronco ◽  
Elena Ochoa ◽  
M. Luján Alvarez ◽  
Ariel Quiroga ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Primary Culture ◽  
Cell Injury ◽  
Cultured Cells ◽  
Male Wistar Rats ◽  
Metabolic Activities ◽  
Stress Oxidative ◽  
Species Production

The aim of this study was to evaluate the influence of partial hepatectomy prior to cell isolation on hepatocytes in vitro. We characterized the possible changes of various stress oxidative parameters within the first 24 h after seeding. Male Wistar rats served as donors. Hepatocytes were isolated by collagenase digestion from either liver of simulated surgery (SH) or from liver 1 h after 70% hepatectomy (PH), and the changes in stress parameters were analyzed after 1, 3, 18, and 24 h in culture. At 24 h, only hepatocytes from PH maintained significantly increased reactive oxygen species production, oxidized glutathione percentage, and Cu/Zn superoxide dismutase and catalase activities. Our results show that hepatocytes suffer significant cell injury as a result of the isolation procedure, but primary cultured cells from SH metabolically recover from this stress after 18 h. After this time, primary culture hepatocytes primed by PH maintain their in vivo-like metabolic activities (increase in both oxidative stress and antioxidant status).

Dependence of the Raman spectra of drug substances upon laser excitation wavelength

2006 ◽  
Vol 37 (1-3) ◽  
pp. 335-341 ◽  
Author(s):  
Fiona C. Thorley ◽  
Kurt J. Baldwin ◽  
David C. Lee ◽  
David N. Batchelder
Keyword(s):  
Raman Spectra ◽  
Laser Excitation ◽  
Excitation Wavelength ◽  
Drug Substances
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