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Oncotarget Apr 2016
Topics: Animals; COS Cells; Humans; Potassium Channels, Voltage-Gated; Stomach Neoplasms
PubMed: 26956055
DOI: 10.18632/oncotarget.7921 -
Scientific Reports Feb 2019Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a...
Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a laser, to produce temporary mechanical deformations of the cell membrane and facilitate the delivery of plasmid DNA into cells. We show that high stress gradients, produced when picosecond laser pulses with a fluence of 100 mJ/cm are absorbed by piezophotonic materials, enable transfection of a plasmid DNA encoding Green Fluorescent Protein (gWizGFP, 3.74 MDa) in COS-7 monkey fibroblast cells with an efficiency of 5% at 20 °C, in 10 minutes. We did not observe significant cytotoxicity under these conditions. Photoacoustic transfection is scalable, affordable, enables nuclear localization and the dosage is easily controlled by the laser parameters.
Topics: Animals; COS Cells; Cell Membrane; Chlorocebus aethiops; Green Fluorescent Proteins; Humans; Photoacoustic Techniques; Plasmids; Transfection
PubMed: 30796229
DOI: 10.1038/s41598-018-37759-1 -
Journal of Visualized Experiments : JoVE Nov 2010Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell...
Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed(1) who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate(2). To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry(1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments(1). The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection(3) in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.
Topics: Animals; COS Cells; Cell Count; Cell Line, Tumor; Chlorocebus aethiops; HEK293 Cells; HeLa Cells; Humans
PubMed: 22158024
DOI: 10.3791/2204 -
Biochimica Et Biophysica Acta.... Oct 2021Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable...
Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable state. FRET based analysis and biochemical data reveal that cytosolic prion (cyPrP) and httQ-103 interact with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leading to few detectable aggregates in GAPDH-over expressing cells.The preventive effect of GAPDH suggests that this abundant and long-lived cytoplasmic protein has an active role in the shielding and maintenance, in soluble form of proteins as heterogeneous as huntingtin and cyPrP.
Topics: Animals; COS Cells; Cell Line; Cell Line, Tumor; Chlorocebus aethiops; Glyceraldehyde-3-Phosphate Dehydrogenases; HeLa Cells; Humans; Protein Aggregates
PubMed: 34144092
DOI: 10.1016/j.bbadis.2021.166202 -
Biophysical Journal Sep 2018Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically...
Reliable interpretation and quantification of cellular features in fluorescence microscopy requires an accurate estimate of microscope resolution. This is typically obtained by measuring the image of a nonbiological proxy for a point-like object, such as a fluorescent bead. Although appropriate for confocal microscopy, bead-based measurements are problematic for stimulated emission depletion microscopy and similar techniques where the resolution depends critically on the choice of fluorophore and acquisition parameters. In this article, we demonstrate that for a known geometry (e.g., tubules), the resolution can be measured in situ by fitting a model that accounts for both the point spread function (PSF) and the fluorophore distribution. To address the problem of coupling between tubule diameter and PSF width, we developed a technique called nested-loop ensemble PSF fitting. This approach enables extraction of the size of cellular features and the PSF width in fixed-cell and live-cell images without relying on beads or precalibration. Nested-loop ensemble PSF fitting accurately recapitulates microtubule diameter from stimulated emission depletion images and can measure the diameter of endoplasmic reticulum tubules in live COS-7 cells. Our algorithm has been implemented as a plugin for the PYthon Microscopy Environment, a freely available and open-source software.
Topics: Animals; COS Cells; Cell Survival; Chlorocebus aethiops; Endoplasmic Reticulum; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Software
PubMed: 30139523
DOI: 10.1016/j.bpj.2018.07.028 -
The Journal of Cell Biology Mar 2014Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane... (Review)
Review
Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle's cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis.
Topics: Animals; COS Cells; Cell Membrane; Chlorocebus aethiops; Energy Metabolism; Fatty Acids; Lipid Metabolism; Metabolic Networks and Pathways; Mice; Phospholipids
PubMed: 24590170
DOI: 10.1083/jcb.201311051 -
Frontiers in Endocrinology 2022Results of previous studies provided evidence for the existence of a functional gonadotropin-inhibitory hormone (GnIH) system in the European sea bass, , which exerted...
Results of previous studies provided evidence for the existence of a functional gonadotropin-inhibitory hormone (GnIH) system in the European sea bass, , which exerted an inhibitory action on the brain-pituitary-gonadal axis of this species. Herein, we further elucidated the intracellular signaling pathways mediating in sea bass GnIH actions and the potential interactions with sea bass kisspeptin (Kiss) signaling. Although GnIH1 and GnIH2 had no effect on basal CRE-luc activity, they significantly decreased forskolin-elicited CRE-luc activity in COS-7 cells transfected with their cognate receptor GnIHR. Moreover, an evident increase in SRE-luc activity was noticed when COS-7 cells expressing GnIHR were challenged with both GnIH peptides, and this stimulatory action was significantly reduced by two inhibitors of the PKC pathway. Notably, GnIH2 antagonized Kiss2-evoked CRE-luc activity in COS-7 cells expressing GnIHR and Kiss2 receptor (Kiss2R). However, GnIH peptides did not alter NFAT-RE-luc activity and ERK phosphorylation levels. These data indicate that sea bass GnIHR signals can be transduced through the PKA and PKC pathways, and GnIH can interfere with kisspeptin actions by reducing its signaling. Our results provide additional evidence for the understanding of signaling pathways activated by GnIH peptides in teleosts, and represent a starting point for the study of interactions with multiple neuroendocrine factors on cell signaling.
Topics: Animals; Bass; COS Cells; Chlorocebus aethiops; Chorionic Gonadotropin; Kisspeptins; Signal Transduction
PubMed: 36051397
DOI: 10.3389/fendo.2022.982246 -
Viruses Dec 2022In relation to the comment by Henriksen and Rinaldo, the authors intend to emphasize that before every experiment with SVGp12 cells they routinely test the cells for the...
Reply to Henriksen, S.; Rinaldo, C.H. Should SVGp12 Be Used for JC Polyomavirus Studies? Comment on "Prezioso et al. COS-7 and SVGp12 Cellular Models to Study JCPyV Replication and MicroRNA Expression after Infection with Archetypal and Rearranged-NCCR Viral Strains. 2022, , 2070".
In relation to the comment by Henriksen and Rinaldo, the authors intend to emphasize that before every experiment with SVGp12 cells they routinely test the cells for the absence of BKPyV contamination. The scientists can state that the SVGp12 cells used in their laboratory were not infected by BKPyV and that their results were also validated on the COS-7 cell line, which is permissive for JCPyV infection. Therefore, the overall findings of the study and its conclusions remain authentic. The authors recommend the necessity of carefully testing SVGp12 cells for BKPyV infection before use or, alternatively, in case of a first purchase; moreover, it is possible to choose different cell lines to avoid running into this unpleasant situation.
Topics: Animals; BK Virus; Chlorocebus aethiops; COS Cells; JC Virus; MicroRNAs
PubMed: 36680133
DOI: 10.3390/v15010093 -
PloS One 2016Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers...
Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).
Topics: Aniline Compounds; Animals; Antineoplastic Agents; Blotting, Western; COS Cells; Cell Cycle; Cell Line; Cell Proliferation; Chlorocebus aethiops; Female; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Neoplasms; Quinazolines; Receptors, Fibroblast Growth Factor
PubMed: 27627808
DOI: 10.1371/journal.pone.0162594 -
Journal of Visualized Experiments : JoVE Nov 2017Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in...
Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61β, and a plasma membrane-localized voltage-gated L-type Ca channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.
Topics: Animals; COS Cells; Chlorocebus aethiops; Image Processing, Computer-Assisted; Mice; Microscopy, Fluorescence; Transfection
PubMed: 29155750
DOI: 10.3791/56239