scholarly journals Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker

Antibodies ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 29
Author(s):  
Stéphanie Charrin ◽  
Roberta Palmulli ◽  
Martine Billard ◽  
Denis Clay ◽  
Claude Boucheix ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Protein A ◽  
Hybridoma Cells ◽  
Late Endosomes ◽  
Electron Microscopy Analysis ◽  
Direct Binding ◽  
Microscopy Analysis ◽  
Igg1 Subclass ◽  
Lysosome Related Organelles ◽  
Limited Dilution

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

scholarly journals Role of Recycling Endosomes and Lysosomes in Dynein-Dependent Entry of Canine Parvovirus

2002 ◽  
Vol 76 (9) ◽  
pp. 4401-4411 ◽  
Author(s):  
Sanna Suikkanen ◽  
Katja Sääjärvi ◽  
Jonna Hirsimäki ◽  
Outi Välilehto ◽  
Hilkka Reunanen ◽  
...  
Keyword(s):  
Canine Parvovirus ◽  
Host Cells ◽  
Coated Pits ◽  
Microtubule Network ◽  
Recycling Endosomes ◽  
Entry Pathway ◽  
Late Endosomes ◽  
Electron Microscopy Analysis ◽  
Nonenveloped Virus ◽  
Microscopy Analysis

ABSTRACT Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.

scholarly journals Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

2020 ◽  
Author(s):  
Asuncion Carmona ◽  
Francesco Porcaro ◽  
Andrea Somogyi ◽  
Stéphane Roudeau ◽  
Florelle Domart ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Protein A ◽  
Uranium Content ◽  
Continuous Exposure ◽  
Analytical Sensitivity ◽  
Iron Storage ◽  
Fetuin A ◽  
Late Endosomes ◽  
Monoaminergic System ◽  
High Analytical Sensitivity

ABSTRACTUranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 μg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 μM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62% of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labelled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and the resulting neurotoxic effects.

Material analysis techniques using the ion mill

1996 ◽  
Vol 54 ◽  
pp. 1034-1035
Author(s):  
J. P. Benedict ◽  
R. M. Anderson ◽  
S. J. Klepeis
Keyword(s):  
Polished Surface ◽  
Surface Material ◽  
Analysis Techniques ◽  
Material Analysis ◽  
Optical Inspection ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Argon Ions ◽  
The Impact ◽  
Scanning Electron

Ion mills equipped with flood guns can perform two important functions in material analysis; they can either remove material or deposit material. The ion mill holder shown in Fig. 1 is used to remove material from the polished surface of a sample for further optical inspection or SEM ( Scanning Electron Microscopy ) analysis. The sample is attached to a pohshing stud type SEM mount and placed in the ion mill holder with the polished surface of the sample pointing straight up, as shown in Fig 2. As the holder is rotating in the ion mill, Argon ions from the flood gun are directed down at the top of the sample. The impact of Argon ions against the surface of the sample causes some of the surface material to leave the sample at a material dependent, nonuniform rate. As a result, the polished surface will begin to develop topography during milling as fast sputtering materials leave behind depressions in the polished surface.

An immunocytochemical study of maternal immunoglobulin localization in the suckling pig intestine

1990 ◽  
Vol 48 (3) ◽  
pp. 922-923
Author(s):  
László G. Kömüves ◽  
Donna S. Turner ◽  
Kathy S. McKee ◽  
Buford L. Nichols ◽  
Julian P. Heath
Keyword(s):  
Sodium Ethoxide ◽  
Intracellular Localization ◽  
Protein A ◽  
Tween 20 ◽  
Intestinal Epithelial ◽  
Immunocytochemical Study ◽  
Fish Skin ◽  
Newborn Piglets ◽  
Rabbit Igg ◽  
Fish Skin Gelatin

In this study we used colloidal gold probes to detect the intracellular localization of colostral immunoglobulins in intestinal epithelial cells of newborn piglets.Tissues were obtained from non-suckled newborn and suckled piglets aged between 1 hour to 1 month. Samples were fixed in 2.5 % glutaraldehyde, osmicated and embedded into Spurr’s resin. Thin (80 nm) sections were etched with 5% sodium ethoxide for 5 min, washed and treated with 4 % sodium-m-periodate in distilled water for 30 min. The sections were then first incubated with blocking buffer (2 % BSA, 0.25 % fish skin gelatin, 0.5 % Tween 20 in 10 mM Trizma buffer, pH=7.4 containing 500 mM NaCl) for 30 min followed by the immunoreagents diluted in the same buffer, 1 hr each. For the detection of pig immunoglobulins a rabbit anti-pig IgG antiserum was used followed by goat anti-rabbit IgG-Au10 or protein A-Au15 probes.

Effects of Various Antioxidant Pretreatment Modalities on Adhesion to Sound and Caries-Affected Dentin: An In Vitro Study

2021 ◽  
pp. 232020682199798
Author(s):  
Beyza Unalan Degirmenci ◽  
Alperen Degirmenci ◽  
Emine Kara
Keyword(s):  
Bond Strength ◽  
In Vitro Study ◽  
Natural Antioxidants ◽  
Microtensile Bond Strength ◽  
Mandibular Molar ◽  
Electron Microscopy Analysis ◽  
Molar Teeth ◽  
Microscopy Analysis ◽  
Two Parameters

Aim: Natural antioxidants were offered as the answer of dentin adhesion issue. The aim of this study is to investigate the effects of proanthocyanidin and lycopene as pretreatment agents on the sound and caries-affected dentin surface on microtensile bond strength and microleakage. Materials and Methods: This study was designed as in vitro because of that 84 mandibular molar teeth were collected. Forty-two of the included teeth were carious teeth, while the other 42 were without caries. Sixty of them were used for microleakage and 24 for microtensile bond strength testing and scanning electron microscopy analysis. The samples were divided into six subgroups randomly according to dentin pretreatments: 5% proanthocyanidin, 5% lycopene, and no antioxidant application. After the restorative procedures, samples were attached to the microtensile tester. Samples were subjected to tensile stress in the load cell until they broke at a speed of 0.5 mm per min. Microtensile bond strength (µTBS) and microleakage test data were analyzed with two-way analysis of variance, Bonferroni correction, and Tamhane’s T2 tests. Results: Two-way variance analysis showed that dentin pretreatment applications, dentin substrate, and the interaction between these two parameters had statistically significant effects on µTBS values ( P < .001). There was no difference between dentin pretreatment applications in terms of microleakage scores ( P > .05). Conclusion: The application of dentin pretreatment with proanthocyanidin is a successful procedure that increases the bond strength in both dentin substrate, while pretreatment with lycopene in caries-affected dentin reduces it.

scholarly journals Antiparasitic Properties of Cardiovascular Agents against Human Intravascular Parasite Schistosoma mansoni

2021 ◽  
Vol 14 (7) ◽  
pp. 686
Author(s):  
Raquel Porto ◽  
Ana C. Mengarda ◽  
Rayssa A. Cajas ◽  
Maria C. Salvadori ◽  
Fernanda S. Teixeira ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Screening Strategy ◽  
Early Infection ◽  
Global Public Health ◽  
Parasitic Worm ◽  
Electron Microscopy Analysis ◽  
Health Significance ◽  
Microscopy Analysis

The intravascular parasitic worm Schistosoma mansoni is a causative agent of schistosomiasis, a disease of great global public health significance. Praziquantel is the only drug available to treat schistosomiasis and there is an urgent demand for new anthelmintic agents. Adopting a phenotypic drug screening strategy, here, we evaluated the antiparasitic properties of 46 commercially available cardiovascular drugs against S. mansoni. From these screenings, we found that amiodarone, telmisartan, propafenone, methyldopa, and doxazosin affected the viability of schistosomes in vitro, with effective concentrations of 50% (EC50) and 90% (EC90) values ranging from 8 to 50 µM. These results were further supported by scanning electron microscopy analysis. Subsequently, the most effective drug (amiodarone) was further tested in a murine model of schistosomiasis for both early and chronic S. mansoni infections using a single oral dose of 400 mg/kg or 100 mg/kg daily for five consecutive days. Amiodarone had a low efficacy in chronic infection, with the worm and egg burden reduction ranging from 10 to 30%. In contrast, amiodarone caused a significant reduction in worm and egg burden in early infection (>50%). Comparatively, treatment with amiodarone is more effective in early infection than praziquantel, demonstrating the potential role of this cardiovascular drug as an antischistosomal agent.

scholarly journals Impact Resistance of Epoxy Composites Reinforced with Amazon Guaruman Fiber: A Brief Report

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2264
Author(s):  
Raphael H. M. Reis ◽  
Fabio C. Garcia Filho ◽  
Larissa F. Nunes ◽  
Veronica S. Candido ◽  
Alisson C. R. Silva ◽  
...  
Keyword(s):  
Fiber Composite ◽  
Impact Resistance ◽  
Fiber Composites ◽  
Epoxy Composites ◽  
Impact Performance ◽  
Electron Microscopy Analysis ◽  
Ballistic Properties ◽  
Microscopy Analysis ◽  
Izod Impact ◽  
The Impact

Fibers extracted from Amazonian plants that have traditionally been used by local communities to produce simple items such as ropes, nets, and rugs, are now recognized as promising composite reinforcements. This is the case for guaruman (Ischinosiphon körn) fiber, which was recently found to present potential mechanical and ballistic properties as 30 vol% reinforcement of epoxy composites. To complement these properties, Izod impact tests are now communicated in this brief report for similar composites with up to 30 vol% of guaruman fibers. A substantial increase in impact resistance, with over than 20 times the absorbed energy for the 30 vol% guaruman fiber composite, was obtained in comparison to neat epoxy. These results were statistically validated by Weibull analysis, ANOVA, and Tukey’s test. Scanning electron microscopy analysis disclosed the mechanisms responsible for the impact performance of the guaruman fiber composites.

scholarly journals Electron microscopy analysis of biofilms produced by Staphylococcus aureus exposed to UV-light on the surface of SnO2 thin films

2021 ◽  
Vol 27 (S1) ◽  
pp. 3168-3170
Author(s):  
Hazel Jaynelle Morales-Rodriguez ◽  
Javier Camarillo-Cisneros ◽  
María Alejandra Favila-Pérez ◽  
Alva Rocío Castillo-González ◽  
Celia María Quiñonez-Flores ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Staphylococcus Aureus ◽  
Uv Light ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Sno2 Thin Films

scholarly journals Focused Ion Beam (FIB) Preparation and Electron Microscopy Analysis of Individual Microbolometer Pixels

2006 ◽  
Vol 12 (S02) ◽  
pp. 1270-1271
Author(s):  
M Olszta ◽  
J Dougherty ◽  
M Horn ◽  
EC Dickey
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2005

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