scholarly journals On the molecular release induced by ultrasound and microbubbles in cells

2021 ◽  
Author(s):  
Farah Hussein
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Negative Pressure ◽  
Membrane Disruption ◽  
Intracellular Uptake ◽  
Recycling Endosomes ◽  
Rpe Cells ◽  
Peak Negative Pressure ◽  
Viable Cells ◽  
The Mean

Ultrasound and microbubble (USMB) enhances intracellular uptake through membrane disruption and endocytosis. This study investigates USMB effects on the molecular release incells through membrane-disruption and exocytosis. Retinal pigmented epithelial (RPE) cells were loaded with Alexa 647-transferrin (Tfn) to mark recycling endosomes, LAMP-1 antibody was used to mark lysosomes, GFP-transfected RPE cells were used to mark cytoplasm, and 7-AAD was used to assess cell viability. USMB exposure was done at 570kPa peak negative pressure for 1min. The mean fluorescent intensities (MFI) of markers were measured using flow cytometry. USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells respectively. LAMP-1 antibody MFI increased by 50% and 15-folds in viable and non-viable cells indicating USMB induced release from lysosomes. Furthermore, Tfn release from recycling endosomes increased by 22% only in viable cells. In conclusion, USMB enhances the molecular release from cytoplasm, lysosomes, and recycling endosomes

scholarly journals On the molecular release induced by ultrasound and microbubbles in cells

2021 ◽  
Author(s):  
Farah Hussein
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Negative Pressure ◽  
Membrane Disruption ◽  
Intracellular Uptake ◽  
Recycling Endosomes ◽  
Rpe Cells ◽  
Peak Negative Pressure ◽  
Viable Cells ◽  
The Mean

Ultrasound and microbubble (USMB) enhances intracellular uptake through membrane disruption and endocytosis. This study investigates USMB effects on the molecular release incells through membrane-disruption and exocytosis. Retinal pigmented epithelial (RPE) cells were loaded with Alexa 647-transferrin (Tfn) to mark recycling endosomes, LAMP-1 antibody was used to mark lysosomes, GFP-transfected RPE cells were used to mark cytoplasm, and 7-AAD was used to assess cell viability. USMB exposure was done at 570kPa peak negative pressure for 1min. The mean fluorescent intensities (MFI) of markers were measured using flow cytometry. USMB induced the release of 19% and 67% of GFP from the cytoplasm in viable and non-viable cells respectively. LAMP-1 antibody MFI increased by 50% and 15-folds in viable and non-viable cells indicating USMB induced release from lysosomes. Furthermore, Tfn release from recycling endosomes increased by 22% only in viable cells. In conclusion, USMB enhances the molecular release from cytoplasm, lysosomes, and recycling endosomes

scholarly journals Immunofluorescence Microscopy and Flow Cytometry Characterization of Chemical Induction of Latent Epstein-Barr Virus

1998 ◽  
Vol 5 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Hal B. Jenson ◽  
George M. Grant ◽  
Yasmin Ench ◽  
Patty Heard ◽  
Charles A. Thomas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Epstein Barr Virus ◽  
Immunofluorescence Microscopy ◽  
Chemical Induction ◽  
Facs Analysis ◽  
Raji Cells ◽  
Barr Virus ◽  
Viable Cells ◽  
Epstein Barr

ABSTRACT The effects of chemical induction of latent Epstein-Barr virus (EBV) with 12-O-tetradecanoyl phorbol-13-acetate (TPA) andn-butyrate on cell viability and induction of latent EBV in Raji and X50-7 B lymphocytes, indicated by expression of the diffuse component of the EBV early antigen (EA-D), were measured by visual immunofluorescence microscopy (of both viable and nonviable cells) and fluorescence-activated cell sorter (FACS) flow cytometry (of viable cells only). Cell viability at 4 days decreased moderately for treated Raji cells (9 to 37%, compared to 55 to 69% for untreated cells) and markedly for X50-7 cells (1-32% compared to 35-44% in untreated cells). The highest EA-D levels in viable cells occurred in Raji cells treated with both TPA and n-butyrate and untreated X50-7 cells. TPA and n-butyrate acted synergistically to induce latent EBV, resulting in increased levels of EA-D production in Raji cells and cell death in X50-7 cells. Methodological differences including the ability to detect antigen in only viable cells by FACS flow cytometry accounted for the higher levels of EA-D observed by FACS analysis compared to the levels observed by immunofluorescence microscopy. FACS analysis may be more objective and reproducible than immunofluorescence microscopy for the detection of EBV induction and also permits viral protein expression to be distinguished in the subpopulation of viable cells.

scholarly journals Mechanisms of Cytotoxicity of Chemical Agents to Giant Cell Tumors: An In Vitro Study

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Achmad Fauzi Kamal ◽  
Akbar Rizki Beni Asdi ◽  
Ahmad Jabir Rahyussalim ◽  
Rio Wikanjaya ◽  
Resda Akhra Syahrani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Giant Cell ◽  
Morphological Changes ◽  
Ethanol Exposure ◽  
Giant Cell Tumors ◽  
Dependent Effect ◽  
Viable Cells ◽  
Chemical Agents

Background. Various chemical agents have been used as an adjuvant treatment for giant cell tumor (GCT). However, the comparative effect of these chemicals remains unclear. Methods. Multinucleated and spindle cells from cultured GCT patients, characterized by Nanog and Oct4 expression with RT-PCR, were directly administered, in vitro, with concentrations of 1%, 3%, and 5% of H2O2 and 75%, 85%, and 95% of ethanol for 10 minutes and concentrations of 0.003%, 0.005%, 0.01%, 0.03%, 0.1%, and 0.3% of H2O2 for 5 minutes and were incubated for 24 hours. Cell morphology, cell viability, and flow cytometry after various concentrations of H2O2 and ethanol exposure were assessed. Results. H2O2 in all concentrations caused loss of cell viability. The number of viable cells after H2O2 exposure was related to the concentration-dependent effect. The initial viable spindle-shaped cell, multinucleated giant cell, and round-epithelioid cell had morphological changes into fragmented nonviable cells after exposure to H2O2. Flow cytometry using Annexin V showed cell death due to necrosis, with the highest concentration amounting to 0.3%. Conclusion. Administering local chemical adjuvants of H2O2 in vitro caused loss of viable GCT cells. The number of viable cells after H2O2 exposure was related to the concentration-dependent effect, whereas reducing concentration of H2O2 may cause loss of viability and morphology of cultured GCT cells with the apoptotic mechanism.

scholarly journals Nanodroplet-mediated catheter-directed sonothrombolysis of retracted blood clots

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Leela Goel ◽  
Huaiyu Wu ◽  
Bohua Zhang ◽  
Jinwook Kim ◽  
Paul A. Dayton ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Negative Pressure ◽  
Boiling Point ◽  
Clot Lysis ◽  
Control Group ◽  
Peak Negative Pressure ◽  
Blood Clots ◽  
Tissue Plasminogen ◽  
First Time

AbstractOne major challenge in current microbubble (MB) and tissue plasminogen activator (tPA)-mediated sonothrombolysis techniques is effectively treating retracted blood clots, owing to the high density and low porosity of retracted clots. Nanodroplets (NDs) have the potential to enhance retracted clot lysis owing to their small size and ability to penetrate into retracted clots to enhance drug delivery. For the first time, we demonstrate that a sub-megahertz, forward-viewing intravascular (FVI) transducer can be used for ND-mediated sonothrombolysis, in vitro. In this study, we determined the minimum peak negative pressure to induce cavitation with low-boiling point phase change nanodroplets and clot lysis. We then compared nanodroplet mediated sonothrombolysis to MB and tPA mediate techniques. The clot lysis as a percent mass decrease in retracted clots was 9 ± 8%, 9 ± 5%, 16 ± 5%, 14 ± 9%, 17 ± 9%, 30 ± 8%, and 40 ± 9% for the control group, tPA alone, tPA + US, MB + US, MB + tPA + US, ND + US, and ND + tPA + US groups, respectively. In retracted blood clots, combined ND- and tPA-mediated sonothrombolysis was able to significantly enhance retracted clot lysis compared with traditional MB and tPA-mediated sonothrombolysis techniques. Combined nanodroplet with tPA-mediated sonothrombolysis may provide a feasible strategy for safely treating retracted clots.

scholarly journals Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Jianghao Gong ◽  
Shangjun Fu ◽  
Zhenghao Zhou
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Protein Level ◽  
Reactive Oxygen ◽  
Octacalcium Phosphate ◽  
Control Group ◽  
Substrate Protein ◽  
Autophagy Inhibitor ◽  
Osteoblast Cell Line

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

scholarly journals Alkannin Inhibits the Development of Ovarian Cancer by Affecting miR-4461

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Yaowen Wang ◽  
Jingfang Zhang ◽  
Feipeng Wang ◽  
Wenping Chen ◽  
Jie Ma ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Cell Viability ◽  
Migration And Invasion ◽  
Flow Cytometry Analysis ◽  
Antibacterial Effects ◽  
Transwell Assay ◽  
Ovarian Cells ◽  
Mrna And Protein Expression ◽  
Inhibit Cell Proliferation

Background. Previous studies have shown that alkannin has anticancer, anti-inflammatory, and antibacterial effects. However, the effect of alkannin in the development of ovarian cancer (OC) remains unknown. Therefore, this study aims to elucidate the function of alkannin in OC progression. Methods. RT-qPCR and western blot analysis were used to measure mRNA and protein expression. Cell viability and metastasis were detected by the CCK-8 assay, flow cytometry analysis, and transwell assay. Results. Alkannin had no cytotoxicity toward normal ovarian cells, but alkannin can inhibit cell proliferation and induce apoptosis in OC cells. In addition, alkannin inhibited cell migration and invasion and blocked EMT in OC. Besides, upregulation of miR-4461 was found in OC tissues and cells, which was regulated by alkannin. More importantly, miR-4461 can inverse the effects of alkannin on cell viability and metastasis in OC cells. Conclusion. Alkannin restrains cell viability, metastasis, and EMT in OC by downregulating miR-4461 expression.

scholarly journals Effect of Temperature and Cell Viability on Uranium Biomineralization by the Uranium Mine Isolate Penicillium simplicissimum

2021 ◽  
Vol 12 ◽  
Author(s):  
Sebastian Schaefer ◽  
Robin Steudtner ◽  
René Hübner ◽  
Evelyn Krawczyk-Bärsch ◽  
Mohamed L. Merroun
Keyword(s):  
Heavy Metal ◽  
Cell Viability ◽  
Laser Fluorescence ◽  
Contaminated Sites ◽  
Effect Of Temperature ◽  
Uranium Mine ◽  
Penicillium Simplicissimum ◽  
Removal Capacity ◽  
Viable Cells ◽  
Dry Biomass

The remediation of heavy-metal-contaminated sites represents a serious environmental problem worldwide. Currently, cost- and time-intensive chemical treatments are usually performed. Bioremediation by heavy-metal-tolerant microorganisms is considered a more eco-friendly and comparatively cheap alternative. The fungus Penicillium simplicissimum KS1, isolated from the flooding water of a former uranium (U) mine in Germany, shows promising U bioremediation potential mainly through biomineralization. The adaption of P. simplicissimum KS1 to heavy-metal-contaminated sites is indicated by an increased U removal capacity of up to 550 mg U per g dry biomass, compared to the non-heavy-metal-exposed P. simplicissimum reference strain DSM 62867 (200 mg U per g dry biomass). In addition, the effect of temperature and cell viability of P. simplicissimum KS1 on U biomineralization was investigated. While viable cells at 30°C removed U mainly extracellularly via metabolism-dependent biomineralization, a decrease in temperature to 4°C or use of dead-autoclaved cells at 30°C revealed increased occurrence of passive biosorption and bioaccumulation, as confirmed by scanning transmission electron microscopy. The precipitated U species were assigned to uranyl phosphates with a structure similar to that of autunite, via cryo-time-resolved laser fluorescence spectroscopy. The major involvement of phosphates in U precipitation by P. simplicissimum KS1 was additionally supported by the observation of increased phosphatase activity for viable cells at 30°C. Furthermore, viable cells actively secreted small molecules, most likely phosphorylated amino acids, which interacted with U in the supernatant and were not detected in experiments with dead-autoclaved cells. Our study provides new insights into the influence of temperature and cell viability on U phosphate biomineralization by fungi, and furthermore highlight the potential use of P. simplicissimum KS1 particularly for U bioremediation purposes.Graphical Abstract

scholarly journals FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

2020 ◽  
Author(s):  
Lydia Horndler ◽  
Pilar Delgado ◽  
Ivaylo Balabanov ◽  
Georgina Cornish ◽  
Miguel Angel Llamas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Correct Identification ◽  
Truncated Form ◽  
Serological Tests ◽  
S Protein ◽  
Human Antibodies ◽  
Degree Of Protection ◽  
Immunoglobulin Isotypes ◽  
Jurkat T Cell ◽  
The Mean

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.

scholarly journals Application of Flow Cytometry to Segregated Kinetic Modeling Based on the Physiological States of Microorganisms

2007 ◽  
Vol 73 (12) ◽  
pp. 3993-4000 ◽  
Author(s):  
Covadonga Quir�s ◽  
M�nica Herrero ◽  
Luis A. Garc�a ◽  
Mario D�az
Keyword(s):  
Flow Cytometry ◽  
Culture Media ◽  
Glucose Consumption ◽  
Viable Cell ◽  
Counting Method ◽  
Batch Cultures ◽  
Viable Cells ◽  
The Status ◽  
Standard Culture

ABSTRACT Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.

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