scholarly journals High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

2019 ◽  
Author(s):  
Gopika SenthilKumar ◽  
Justin H. Skiba ◽  
Randall J. Kimple
Keyword(s):  
Acridine Orange ◽  
High Throughput ◽  
Late Stage ◽  
Image Cytometry ◽  
Alternative Methods ◽  
Quantitative Detection ◽  
Autophagic Flux ◽  
Objective Method ◽  
Acridine Orange Staining ◽  
Transmission Electron

AbstractQuantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevent accurate quantification. We report a high-throughput, inexpensive, reliable, and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.Methods summaryA high-throughput, inexpensive, reliable, and objective method for studying both basal autophagy and autophagic flux is reported. This approach uses acridine orange staining of late-stage autophagy and image cytometry to quantify autophagy.

scholarly journals High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry

BioTechniques ◽  
2019 ◽  
Vol 67 (2) ◽  
pp. 70-73 ◽  
Author(s):  
Gopika SenthilKumar ◽  
Justin H Skiba ◽  
Randall J Kimple
Keyword(s):  
High Throughput ◽  
Image Cytometry ◽  
Quantitative Detection ◽  
Autophagic Flux

scholarly journals NOS2 inhibitor 1400W Induces Autophagic Flux and Influences Extracellular Vesicle Profile in Human Glioblastoma U87MG Cell Line

2019 ◽  
Vol 20 (12) ◽  
pp. 3010 ◽  
Author(s):  
Paola Palumbo ◽  
Francesca Lombardi ◽  
Francesca Rosaria Augello ◽  
Ilaria Giusti ◽  
Sabino Luzzi ◽  
...  
Keyword(s):  
Extracellular Vesicle ◽  
Acid Sphingomyelinase ◽  
Cell Counting ◽  
Autophagic Flux ◽  
U87mg Cell ◽  
Cell Growth And Migration ◽  
Acridine Orange Staining ◽  
Transmission Electron ◽  
And Migration ◽  
Nitric Oxide Synthase 2

The relevance of nitric oxide synthase 2 (NOS2) as a prognostic factor in Glioblastoma Multiforme (GBM) malignancy is emerging. We analyzed the effect of NOS2 inhibitor 1400W on the autophagic flux and extracellular vesicle (EV) secretion in U87MG glioma cells. The effects of glioma stem cells (GSC)-derived EVs on adherent U87MG were evaluated. Cell proliferation and migration were examined while using Cell Counting Kit-8 assay (CCK-8) and scratch wound healing assay. Cell cycle profile and apoptosis were analyzed by flow cytometry. Autophagy-associated acidic vesicular organelles were detected and quantified by acridine orange staining. The number and size of EVs were assessed by nanoparticle tracking analysis. EV ultrastructure was verified by transmission electron microscopy (TEM). WB was used to analyze protein expression and acid sphingomyelinase was determined through ceramide levels. 1400W induced autophagy and EV secretion in both adherent U87MG and GSCs. EVs secreted by 1400W-treated GSC, but not those from untreated cells, were able to inhibit adherent U87MG cell growth and migration while also inducing a relevant level of autophagy. The hypothesis of NOS2 expression as GBM profile marker or interesting therapeutic target is supported by our findings. Autophagy and EV release following treatment with the NOS2 inhibitor could represent useful elements to better understand the complex biomolecular frame of GBM.

Gadolinium Oxide Nanoparticles Enhance the Cytotoxicity of Chemotherapeutic Drugs by Blocking Autophagic Flux in Human Ovarian Cancer Cells

2020 ◽  
Vol 16 ◽  
Author(s):  
Zhixiong Xie ◽  
Tianyu Zhang ◽  
Cheng Zhong
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Hela Cells ◽  
Late Stage ◽  
Ovarian Cancer Cells ◽  
Oxide Nanoparticles ◽  
Autophagic Flux ◽  
Human Ovarian Cancer ◽  
Chemotherapeutic Drugs ◽  
Chemotherapy Drugs

Background: During chemotherapy, drugs can damage cancer cells’ DNA and cytomembrane structure, and then induce cell death. However, autophagy can increase the chemotherapy resistance of cancer cells, reducing the effect of chemotherapy. Objective: To block the autophagic flux in cancer cells, it is vital to enhance the anti-cancer efficacy of chemotherapy drugs; for this purpose, we test the gadolinium oxide nanoparticles (Gd2O3 NPs)’ effect on autophagy. Methods: The cytotoxicity of Gd2O3 NPs on HeLa cells was evaluated by a (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then, monodasylcadaverine staining, immunofluorescence, immunoblot and apoptosis assay were conducted to evaluate the effect of Gd2O3 NPs on autophagy and efficacy of chemotherapy drugs in human ovarian cancer cells. Results: We found that Gd2O3 NPs, which have great potential for use as a contrast agent in magnetic resonance imaging, could block the late stage of autophagic flux in a dose-dependent manner and then cause autophagosome accumulation in HeLa cells. When co-treated with 8 μg/mL Gd2O3 NPs and 5 μg/mL cisplatin, the number of dead HeLa cells increased by about 20% compared with cisplatin alone. We observed the same phenomenon in cisplatin-resistant COC1/DDP cells. Conclusion: We conclude that Gd2O3 NPs can block the late stage of autophagic flux and enhance the cytotoxicity of chemotherapeutic drugs in human ovarian cancer cells. Thus, the nanoparticles have significant potential for use in both diagnosis and therapy of solid tumor.

scholarly journals High-throughput viral microneutralization method for feline coronavirus using image cytometry

2020 ◽  
Vol 286 ◽  
pp. 113979
Author(s):  
Morgan Pearson ◽  
Alora LaVoy ◽  
Leo Li-Ying Chan ◽  
Gregg A. Dean
Keyword(s):  
High Throughput ◽  
Image Cytometry ◽  
Feline Coronavirus

STUDIES ON THE BIOLOGICAL PROPERTIES AND CLASSIFICATION OF SV4 VIRUS

1965 ◽  
Vol 11 (2) ◽  
pp. 325-335 ◽  
Author(s):  
S. A. Sattar ◽  
K. R. Rozee
Keyword(s):  
Electron Microscope ◽  
Rhesus Monkey ◽  
Red Blood Cells ◽  
Acridine Orange ◽  
Blood Cells ◽  
Magnesium Chloride ◽  
Fluorescent Microscopy ◽  
Biological Properties ◽  
Acridine Orange Staining

Cytopathic changes in LLC-MK2 cells infected with SV4 virus, observed with the electron microscope and using acridine orange staining and fluorescent microscopy, have been shown to be similar to that caused by picornaviruses and members of the Columbia-SK virus group. The virus was found to be stabilized against heat in the presence of molar magnesium chloride, and to be stable at pH 3.5. The virus was non-pathogenic for suckling mice, failed to agglutinate sheep and human "O" red blood cells, but agglutinated rhesus monkey erythrocytes at 4 °C. On the basis of these properties and those already known, it was suggested that SV4 virus be placed in the group Enteroviruses of lower animals.

scholarly journals CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS

1962 ◽  
Vol 15 (3) ◽  
pp. 535-540 ◽  
Author(s):  
M. Rabinovitch ◽  
W. Plaut
Keyword(s):  
Nucleic Acid ◽  
Dna Synthesis ◽  
Acridine Orange ◽  
Particle Number ◽  
Amoeba Proteus ◽  
Acridine Orange Staining ◽  
Particle Population ◽  
Cell Age ◽  
Cytoplasmic Dna ◽  
Number Of Particles

Nucleic acid-containing particles in the cytoplasm of Amoeba proteus (cf. reference 1) were counted after acridine orange staining. The number of particles per ameba was found to be correlated with cell age and size. Fresh daughters had a mean particle number of 5400, whereas predivision amebae contained around 11,000 particles. Amebae from two other strains contained similar particles. The particles were found to be clustered in fasted cells and redispersed after feeding. A marked increase in the particle population was noted in anucleate fragments. These results, together with those previously presented, suggest that the particles multiply intracellularly. Their nature and their relationship to previous work on nucleic acid labeling in Amoeba are discussed.

The fluorescent staining of a mycobacteriophage (C2) nucleic acid

1971 ◽  
Vol 17 (2) ◽  
pp. 171-174 ◽  
Author(s):  
Jose Menezes
Keyword(s):  
Nucleic Acid ◽  
Acridine Orange ◽  
Staining Technique ◽  
Fluorescent Staining ◽  
Green Fluorescence ◽  
Acridine Orange Staining ◽  
Fluorescence Characteristic ◽  
Phage Dna ◽  
Acid Dnase

By using the acridine orange staining technique a green fluorescence, characteristic of double-stranded nucleic acid, can be observed with purified preparations of mycobacteriophage C2 and its extracted nucleic acid. DNAse-treated samples do not show this fluorescence, which leads to the conclusion that this fluorescence is associated with phage DNA. Examination of preparations of phage grown in the presence of acridine orange supported these results.

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