calcein-am and thiazolyl-blue

Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (10

Topics: Animals; Anti-Infective Agents; Body Fluids; Cell Line; Cell Survival; COVID-19; Electrochemistry; Fluoresceins; Humans; Hydrogen Peroxide; Kinetics; Nanoparticles; Propidium; SARS-CoV-2; Swine; Temperature; Tetrazolium Salts; Textiles; Thiazoles; Virion; Wound Healing

The purpose of this in vitro study was to compare the biocompatibility of a novel formulation of a silicone-based endodontic sealer GuttaFlow 2 (GF2; Coltène/Whaledent, Langenau, Germany) with the original (GFO) and fast-set (GFF) formulations of GuttaFlow and with an epoxy resin sealer, AHPlus Jet (AH+J; Dentsply, York, PA).. Sealers were set into 3 × 5.5 mm discs. Cell culture media was used to extract leachable products at 24 hours and 1, 2, and 4 weeks. Primary human periodontal ligament fibroblasts were incubated with sealer elutes for 24 hours and evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the calcein AM assay. Cell attachment was evaluated on set sealer that was either rinsed or unrinsed with cell media for 1 week. Statistical analysis was performed using the Student t test.. Both calcein and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays revealed that periodontal ligament cell viability was reduced on AH+J at 1, 2, and 4 weeks compared with all GuttaFlow sealers. There were no differences in cell viability between the GuttaFlow samples, and all displayed high rates of cell survival at all time periods. After 2 hours, cell attachment to the rinsed GFO and GFF samples exceeded the control, and at 24 hours cell attachment on all GuttaFlow samples exceeded the control. AH+J sealers supported significantly less cell attachment when compared with all GuttaFlow sealers. Cell attachment to set sealers showed better cell attachment when rinsed compared with unrinsed.. GuttaFlow sealers were more biocompatible than AHJ in vitro. The novel GF2 displayed comparable biocompatibility with GFF and GFO.

Topics: Biocompatible Materials; Cell Adhesion; Cell Culture Techniques; Cell Survival; Cells, Cultured; Coloring Agents; Culture Media; Dimethylpolysiloxanes; Drug Combinations; Epoxy Resins; Fibroblasts; Fluoresceins; Fluorescent Dyes; Gutta-Percha; Humans; Humidity; Materials Testing; Periodontal Ligament; Root Canal Filling Materials; Temperature; Tetrazolium Salts; Thiazoles; Time Factors

Ultrasound (US) and microbubbles can be used to facilitate cellular uptake of drugs through a cavitationinduced enhancement of cell membrane permeability. The mechanism is, however, still incompletely understood. A direct contact between microbubbles and cell membrane is thought to be essential to create membrane perturbations lasting from seconds to minutes after US exposure of the cells. A recent study showed that the effect may even last up to 8 h after cavitation (with residual permeability up to 24 h after cavitation). In view of possible membrane damage, the purpose of this study was to further investigate the evolution of cell viability in the range of the 24-h temporal window. Furthermore, a description of the functional changes in tumor cells after US exposure was initiated to obtain a better understanding of the mechanism of membrane perturbation after sonication with microbubbles. Our results suggest that US does not reduce cell viability up to 24 h post-exposure. However, a perturbation of the entire cell population exposed to US was observed in terms of enzymatic activity and characteristics of the mitochondrial membrane. Furthermore, we demonstrated that US cavitation induces a transient loss of cell membrane asymmetry, resulting in phosphatidylserine exposure in the outer leaflet of the cell membrane.

Topics: Acridine Orange; Animals; Annexin A5; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Survival; Fluoresceins; Fluorescent Dyes; Glioma; Microbubbles; Microscopy, Fluorescence; Rats; Sonication; Tetrazolium Salts; Thiazoles

Single-walled carbon nanotubes (SWCNT), fullerenes (C(60)), carbon black (CB), nC(60), and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C(60), nC(60), and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96 AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue (CTB), CytoTox Onetrade mark (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R(2) value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment.

Topics: Cell Line; Cell Survival; Drug Evaluation, Preclinical; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Keratinocytes; Light; Microscopy, Electron, Transmission; Nanoparticles; Oxazines; Quantum Dots; Scattering, Radiation; Spectrophotometry, Ultraviolet; Tetrazolium Salts; Thiazoles; Trypan Blue; Xanthenes

A series of 4,6-dimethoxyaurones were synthesized by reacting 4,6-dimethoxybenzofuran-3(2H)-one with various benzaldehydes in a base-catalyzed aldol reaction. A Z configuration was assigned to the aurones based on spectroscopic and crystallographic data. The aurones were tested for their ability to modulate ABCG2 (breast cancer resistance protein)-mediated multidrug resistance in vitro. Several members (0.5 microM) increased the accumulation of mitoxantrone (MX) in human breast cancer cells (MDA-MB-231) transfected with ABCG2 and re-sensitized these cells to the cytotoxic effects of MX. In the re-sensitization assay, aurones at 0.5 microM reduced the resistance of the transfected cells to MX to just twice that of the parental cells, exceeding fumitremorgin C (FTC) tested at the same concentration. The aurones (10 microM) also increased calcein-AM accumulation in MDCKII/MDR1 cells that were transfected with ABCB1 (P-glycoprotein), at levels comparable to verapamil tested at the same concentration. Structure-activity analysis showed that substitution of the benzylidene ring B of the aurone template was less important for ABCG2 inhibition, with little variation in activity noted for compounds with an unsubstituted ring B or one that was substituted. In contrast, substitution of ring B gave rise to better inhibitors of ABCB1. A preference for the 3' position of ring B was noted. There was also some indication from the data that aurones with good ABCG2 inhibitory activity were poor ABCB1 inhibitors and vice versa, but further confirmation would be required. Limited antiproliferative activity (>70% cell survival) was observed for many aurones on four different cell lines. Thus, functionalized 4,6-dimethoxyaurones are promising ABCG2 inhibitors that combine good activity at submicromolar concentrations with limited antiproliferative activity.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzofurans; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cell Survival; Crystallography, X-Ray; DNA, Complementary; Dogs; Drug Resistance, Neoplasm; Female; Flow Cytometry; Fluoresceins; Humans; Indicators and Reagents; Mitoxantrone; Models, Molecular; Neoplasm Proteins; Structure-Activity Relationship; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

The objective of this study was to investigate whether P-glycoprotein (P-gp) functional expression in intestinal cells is modified after long-term exposure to the food contaminant cadmium (Cd). The Caco-2 cell line, clone TC7, was first validated as a cellular model for long-term exposure to cadmium. Cytotoxicity tests after acute exposure of 24 h showed a significant concentration-dependent decrease in cellular viability at cadmium levels higher than 10 microM and led us to select the cadmium ranges for long-term exposure: 1, 5, and 10 microM. Intestinal cells were exposed to these cadmium concentrations for four consecutive weeks without inducing DNA condensation or fragmentation. In the second part of this work, we studied the functional expression of the drug efflux pump multidrug resistance P-glycoprotein after long-term exposure to cadmium by immunoblotting with the monoclonal antibody F4 and measurement of calcein-AM+/-the P-gp inhibitor verapamil. Western blot analysis with the F4 antibody detected a single band of 170 to 180 kDa which is the size previously reported for P-gp. Calcein-AM assay showed that four weeks exposure of intestinal cells to 1, 5, and 10 microM Cd increased P-gp functional expression in proportion to the Cd concentration.

Topics: ATP Binding Cassette Transporter, Subfamily B; Benzimidazoles; Blotting, Western; Caco-2 Cells; Cadmium Poisoning; Calcium Channel Blockers; Cell Survival; DNA; Drug Resistance, Multiple; Electrophoresis, Agar Gel; Fluoresceins; Fluorescent Dyes; Humans; L-Lactate Dehydrogenase; Neutral Red; Tetrazolium Salts; Thiazoles; Verapamil

Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness.. New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay.. Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only.. This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.

Topics: Animals; Biological Assay; Cell Survival; Chondroitin Sulfates; Coloring Agents; Complex Mixtures; Cornea; Corneal Transplantation; Culture Media, Serum-Free; Dextrans; Ethidium; Female; Fluoresceins; Fluorescent Dyes; Gentamicins; Intercalating Agents; Male; Microscopy, Confocal; Microscopy, Fluorescence; Rabbits; Tetrazolium Salts; Thiazoles; Tissue Preservation