cell viability assay
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2022 ◽  
Vol 23 (2) ◽  
pp. 901
Author(s):  
Zikai Liu ◽  
Qing Cheng ◽  
Xiaoli Ma ◽  
Mingke Song

The role of calcium ion (Ca2+) signaling in tumorigenicity has received increasing attention in melanoma research. Previous Ca2+ signaling studies focused on Ca2+ entry routes, but rarely explored the role of Ca2+ extrusion. Functioning of the Na+/Ca2+ exchanger (NCX) on the plasma membrane is the major way of Ca2+ extrusion, but very few associations between NCX and melanoma have been reported. Here, we explored whether pharmacological modulation of the NCX could suppress melanoma and promise new therapeutic strategies. Methods included cell viability assay, Ca2+ imaging, immunoblotting, and cell death analysis. The NCX inhibitors SN-6 and YM-244769 were used to selectively block reverse operation of the NCX. Bepridil, KB-R7943, and CB-DMB blocked either reverse or forward NCX operation. We found that blocking the reverse NCX with SN-6 or YM-244769 (5–100 μM) did not affect melanoma cells or increase cytosolic Ca2+. Bepridil, KB-R7943, and CB-DMB all significantly suppressed melanoma cells with IC50 values of 3–20 μM. Bepridil and KB-R7943 elevated intracellular Ca2+ level of melanoma. Bepridil-induced melanoma cell death came from cell cycle arrest and enhanced apoptosis, which were all attenuated by the Ca2+ chelator BAPTA-AM. As compared with melanoma, normal melanocytes had lower NCX1 expression and were less sensitive to the cytotoxicity of bepridil. In conclusion, blockade of the forward but not the reverse NCX leads to Ca2+-related cell death in melanoma and the NCX is a potential drug target for cancer therapy.


2022 ◽  
Vol 8 (1) ◽  
Author(s):  
Cheol Ho Park ◽  
Bin Lee ◽  
Myeonggil Han ◽  
Woo Joong Rhee ◽  
Man Sup Kwak ◽  
...  

AbstractSodium-glucose cotransporter 2 inhibitors, which are recently introduced as glucose-lowering agents, improve cardiovascular and renal outcomes in patients with diabetes mellitus. These drugs also have beneficial effects in various kidney disease models. However, the effect of SGLT2 inhibitors on cisplatin-induced acute kidney injury (AKI) and their mechanism of action need to be elucidated. In this study, we investigated whether canagliflozin protects against cisplatin-induced AKI, depending on adenosine monophosphate-activated protein kinase (AMPK) activation and following induction of autophagy. In the experiments using the HK-2 cell line, cell viability assay and molecular analysis revealed that canagliflozin protected renal proximal tubular cells from cisplatin, whereas addition of chloroquine or compound C abolished the protective effect of canagliflozin. In the mouse model of cisplatin-induced AKI, canagliflozin protected mice from cisplatin-induced AKI. However, treatment with chloroquine or compound C in addition to administration of cisplatin and canagliflozin eliminated the protective effect of canagliflozin. Collectively, these findings indicate that canagliflozin protects against cisplatin-induced AKI by activating AMPK and autophagy in renal proximal tubular cells.


2022 ◽  
Author(s):  
Jing Wu ◽  
zhonghao li ◽  
xiaoke dong ◽  
siyuan yuan ◽  
jinmin liu ◽  
...  

Abstract Background: Acute ischemic stroke (AIS) and following reperfusion therapy-induced cerebral ischemia reperfusion (I/R) injury have been recognized as an important subject of cerebrovascular disease with high mortality. Oxidative stress is an important pathological process of cerebral I/R injury. microRNA-19a (miR-19a) is involved in I/R. As the organ protectant agent, Shenmai Injection (SMI) is widely used in the clinical treatment of cerebral infarction. Purpose: This study aims to explore whether SMI can reduce oxidative stress by regulating miR-19a, thereby treating I/R injury. Methods: The oxidative stress state of PC12 cells was induced by H2O2, and then the cells were cultured with SMI. The therapeutic effect of SMI was evaluated by detecting cellular superoxide dismutase (SOD), malondialdehyde (MDA) and other oxidative markers with the kit. Western blot, PCR, immunofluorescence and other techniques were used to elucidate the potential mechanism of SMI. Results: Cell viability assay results showed that SMI could improve the viability of PC12 cells stimulated by H2O2. Compared with the H2O2 group, after SMI treatment, the contents of MDA and reactive oxygen species (ROS) were significantly reduced, while the activity of SOD was significantly increased, and SMI could reduce apoptosis by increasing the content of adenosine 5'-triphosphate (ATP) in cells and enhancing the mitochondrial membrane potential (∆Ψm). Western blot and qRT-PCR results showed that these effects were partially achieved through the AMPK/Sirt1/PGC-1α pathway. The level of miR-19a was significantly increased in H2O2 group, and SMI could protect the cells by reducing miR-19a. Further investigated the target of miR-19a, and transfected cells with miR-19a mimic and inhibitor respectively. We found that AdipoR2 was a direct target of miR-19a, and miR-19a could inhibit AdipoR2/PI3K/Akt/mTOR pathway. Conclusion:SMI can activate AMPK/Sirt1/PGC-1α and AdipoR2/PI3K/Akt/mTOR pathways by reducing miR-19a levels, and protect PC12 cells stimulated by H2O2.


2022 ◽  
Author(s):  
Aleksandar Radivoievych ◽  
Benjamin Kolp ◽  
Sergii Grebinyk ◽  
Svitlana Prylutska ◽  
Uwe Ritter ◽  
...  

Abstract The acoustic pressure waves of ultrasound (US) penetrate biological tissues deeper than light. Another important feature of US its potential to generate light emission within the excited medium termed sonoluminescence. This promoted the idea of its use as an alternative energy source for photosensitizer excitation. Pristine C60 fullerene (C60), an excellent photosensitizer, was explored in the frame of cancer sonodynamic therapy (SDT). For that purpose, we analyzed C60 effects on human cervix carcinoma HeLa cells in combination with a low intensity US treatment. The time-dependent accumulation of C60 in HeLa cells reached its maximum at 24 h (800 ± 66 ng / 106 cells). Half of extranuclear C60 localized within mitochondria. The efficiency of C60 nanostructure’s sonoexcitation with 1 MHz US was tested with cell viability assay. A significant proapoptotic sonotoxic effect was found for HeLa cells. C60’s ability to induce apoptosis of carcinoma cells after sonoexcitation with US provides a promising novel approach for cancer treatment.


2021 ◽  
Vol 22 (24) ◽  
pp. 13557
Author(s):  
Mariella Rosalia ◽  
Priusha Ravipati ◽  
Pietro Grisoli ◽  
Rossella Dorati ◽  
Ida Genta ◽  
...  

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/wPLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 μm and its internal diameter was 2.61 ± 0.05 mm. The graft’s antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.


2021 ◽  
Author(s):  
Graham Anderson ◽  
Andrew McLeod ◽  
Pierre Bagnaninchi ◽  
Baljean Dhillon

The role of ultraviolet radiation (UVR) exposure in the pathology of age-related macular degeneration (AMD) has been debated for decades with epidemiological evidence failing to find a clear consensus for or against it playing a role. A key reason for this is a lack of foundational research into the response of living retinal tissue to UVR in regard to AMD-specific parameters of tissue function. We, therefore, explored the response of cultured retinal pigmented epithelium (RPE), the loss of which heralds advanced AMD, to specific wavelengths of UVR across the UV-B and UV-A bands found in natural sunlight. Using a bespoke in vitro UVR exposure apparatus coupled with bandpass filters we exposed the immortalised RPE cell line, ARPE-19, to 10nm bands of UVR between 290 and 405nm. Physical cell dynamics were assessed during exposure in cells cultured upon specialist electrode culture plates which allow for continuous, non-invasive electrostatic interrogation of key cell parameters during exposure such as monolayer coverage and tight-junction integrity. UVR exposures were also utilised to quantify wavelength-specific effects using a rapid cell viability assay and a phenotypic profiling assay which was leveraged to simultaneously quantify intracellular reactive oxygen species (ROS), nuclear morphology, mitochondrial stress, epithelial integrity and cell viability as part of a phenotypic profiling approach to quantifying the effects of UVR. Electrical impedance assessment revealed unforeseen detrimental effects of UV-A, beginning at 350nm, alongside previously demonstrated UV-B impacts. Cell viability analysis also highlighted increased effects at 350nm as well as 380nm. Effects at 350nm were further substantiated by high content image analysis which highlighted increased mitochondrial dysfunction and oxidative stress. We conclude that ARPE-19 cells exhibit a previously uncharacterised sensitivity to UV-A radiation, specifically at 350nm and somewhat less at 380nm. If upheld in vivo, such sensitivity will have impacts upon geoepidemiological risk scoring of AMD.


Toxics ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 348
Author(s):  
Raúl Bonne Hernández ◽  
Nadja C. de Souza-Pinto ◽  
Jos Kleinjans ◽  
Marcel van Herwijnen ◽  
Jolanda Piepers ◽  
...  

Manganese (Mn) is an important element; yet acute and/or chronic exposure to this metal has been linked to neurotoxicity and neurodegenerative illnesses such as Parkinson’s disease and others via an unknown mechanism. To better understand it, we exposed a human neuroblastoma cell model (SH-SY5Y) to two Mn chemical species, MnCl2 and Citrate of Mn(II) (0–2000 µM), followed by a cell viability assay, transcriptomics, and bioinformatics. Even though these cells have been chemically and genetically modified, which may limit the significance of our findings, we discovered that by using RA-differentiated cells instead of undifferentiated SH-SY5Y cell line, both chemical species induce a similar toxicity, potentially governed by disruption of protein metabolism, with some differences. The MnCl2 altered amino acid metabolism, which affects RNA metabolism and protein synthesis. Citrate of Mn(II), however, inhibited the E3 ubiquitin ligases–target protein degradation pathway, which can lead to the buildup of damaged/unfolded proteins, consistent with histone modification. Finally, we discovered that Mn(II)-induced cytotoxicity in RA-SH-SY5Y cells shared 84 percent of the pathways involved in neurodegenerative diseases.


2021 ◽  
Author(s):  
◽  
Rossella Nicolai

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells.  The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell.  Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>


2021 ◽  
Author(s):  
◽  
Rossella Nicolai

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells.  The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell.  Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>


2021 ◽  
Vol 9 (12) ◽  
pp. 2516
Author(s):  
Javiera Ortiz-Severín ◽  
Julia I. Tandberg ◽  
Hanne C. Winther-Larsen ◽  
Francisco P. Chávez ◽  
Verónica Cambiazo

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen.


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