Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death

2003 ◽  
Vol 284 (4) ◽  
pp. C1083-C1089 ◽  
Author(s):  
Martin M. Knight ◽  
Susan R. Roberts ◽  
David A. Lee ◽  
Dan L. Bader
Keyword(s):  
Cell Death ◽  
Confocal Microscopy ◽  
Live Cell Imaging ◽  
Laser Excitation ◽  
Fluorescent Microscopy ◽  
Confocal Imaging ◽  
Viable Cells ◽  
Numerous Cell ◽  
Cell Processes ◽  
Microscopy Techniques

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2+ transients and oscillations. Decreasing the power of the laser light decreased the percentage of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was ∼90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.

scholarly journals Live-Cell Imaging of Caspase Activation for High-Content Screening

2009 ◽  
Vol 14 (8) ◽  
pp. 956-969 ◽  
Author(s):  
Christophe Antczak ◽  
Toshimitsu Takagi ◽  
Christina N. Ramirez ◽  
Constantin Radu ◽  
Hakim Djaballah
Keyword(s):  
Cell Death ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Exposure Period ◽  
Live Cells ◽  
Assay Method ◽  
Caspase Activation ◽  
Fluorogenic Substrate ◽  
Automated Imaging ◽  
First Time

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488™ fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells. ( Journal of Biomolecular Screening 2009:956-969)

scholarly journals BRCa1 participates in XIST RNa localization on inactive x chromosome

2019 ◽  
Vol 18 (2) ◽  
pp. 21-26
Author(s):  
E. A. Shestakova ◽  
T. A. Bogush
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Confocal Microscopy ◽  
X Chromosome ◽  
Fluorescent Microscopy ◽  
S Phase ◽  
Immunofluorescent Staining ◽  
Heterochromatin Protein ◽  
Inactive X Chromosome ◽  
Xist Rna

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions. 

scholarly journals Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy

2020 ◽  
Vol 98 (5) ◽  
pp. 612-623
Author(s):  
Adam Tepperman ◽  
David Jiao Zheng ◽  
Maria Abou Taka ◽  
Angela Vrieze ◽  
Austin Le Lam ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Imaging Modalities ◽  
Experimental Conditions ◽  
Microscopy Imaging ◽  
Glass Coverslip ◽  
Multiple Imaging ◽  
Microscopy Techniques

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires the provision of appropriate experimental conditions in a format that meets the optical requirements of the microscope. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized, live-cell imaging chambers that are compatible with a range of imaging modalities, including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and that supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting microfluidic devices made of polydimethylsiloxane. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach for producing imaging chambers that are compatible with modern microscopy techniques.

Histamine-induced protein leakage in hypertensive rats: inhibition by verapamil

1986 ◽  
Vol 250 (2) ◽  
pp. H284-H290 ◽  
Author(s):  
F. N. Miller ◽  
I. G. Joshua ◽  
J. T. Fleming ◽  
N. Parekh
Keyword(s):  
Fluorescent Microscopy ◽  
Fluorescein Isothiocyanate ◽  
Calcium Entry Blocker ◽  
Hypertensive Rats ◽  
Rat Serum ◽  
Protein Leakage ◽  
Hypertensive Rat ◽  
Increased Sensitivity ◽  
Microscopy Techniques

Hypertension has been associated with an enhanced transport of macromolecules from the vasculature to the interstitium. The first objective of this study was to determine if, under control conditions, there is an enhanced leakage of macromolecules from the cremaster vasculature of the hypertensive rat. The second objective was to determine if the response to a mediator of macromolecular leakage (histamine) was altered in the renovascular hypertensive rat. A third objective was to determine if a calcium entry blocker, verapamil, could inhibit histamine-induced leakage and, if so, was the sensitivity to verapamil different in the renovascular hypertensive rat. Rats were anesthetized with pentobarbital, and the cremaster preparation was used for in vivo television microscopy studies. Fluorescein isothiocyanate was tagged to rat serum albumin (FITC-RSA), and the leakage of this albumin from the vasculature to the interstitium was quantitated by the use of fluorescent microscopy techniques. There was no difference during control conditions in macromolecular leakage between the normotensive and hypertensive rats. However, histamine induced a greater leakage in the renovascular hypertensive rat than in the normotensive controls. In addition, verapamil, in the presence of normal calcium levels, inhibited the histamine-induced leakage in the hypertensive rats but not in the normotensive controls. These data suggest that enhanced macromolecular leakage during hypertension may be due to an increased sensitivity to mediators of protein leakage. These agents may produce protein leakage by enhancing entry of extracellular calcium into endothelial cells.

scholarly journals Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gillian E. Conway ◽  
Zhonglei He ◽  
Ana Lacramioara Hutanu ◽  
George Paul Cribaro ◽  
Eline Manaloto ◽  
...  
Keyword(s):  
Cell Death ◽  
Glioblastoma Multiforme ◽  
Plasma Treatment ◽  
Acridine Orange ◽  
Molecular Mechanisms ◽  
Confocal Imaging ◽  
Atmospheric Plasma ◽  
Cold Atmospheric Plasma ◽  
Rapid Accumulation ◽  
Lysosomal Accumulation

Abstract Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Quantitative Laser Scanning Confocal Autofluorescence Microscopy (Lscam) of Normal and Premalignant Colonic Tissues

1997 ◽  
Vol 3 (S2) ◽  
pp. 25-26
Author(s):  
Hsing-Wen Wang ◽  
Joseph Willis ◽  
Michael V. Sivak ◽  
Joseph A. Izatt
Keyword(s):  
Laser Scanning ◽  
Laser Excitation ◽  
Argon Laser ◽  
Confocal Imaging ◽  
Premalignant Lesions ◽  
Tubular Adenoma ◽  
Normal Colon ◽  
Ultraviolet Excitation ◽  
Lif Spectroscopy

Laser-induced fluorescence (LIF) spectroscopy of tissues has been proposed as an adjunct to endoscopy for in vivo gastrointestinal (GI) diagnosis of premalignant lesions. In previous studies, under ultraviolet excitation (350-370 nm), sources of LIF signal differences in normal, and premalignant colonie tissues have been identified including 1) changes in gross tissue morphology, 2) increased hemoglobin absorption in adenomatous tissues, and 3) increased red fluorescence in dysplastic cells. However, many questions remain regarding the specific origins and biochemical correlates of GI tissue autofluorescence.We report on a study of quantitative ultraviolet LIF spectral microscopy in human colonic tissues using a laser scanning confocal microscope with argon laser excitation 351-364 nm. Frozen sections (6μm) of normal colon (n=10) and tubular adenoma (n=8) were prepared from fresh surgical resection specimens using a previously published protocol. To identify histological components accurately, LSCAM slides were alcohol fixed and H&E histologically stained following confocal imaging.

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