Measurement of Autophagy by Flow Cytometry

Warnes, Gary

doi:10.1002/0471142956.cy0945s68

Cited by 16 publications

(15 citation statements)

References 33 publications

(69 reference statements)

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“…Considering that the increase of LC3II/LC3I ratio was detected in whole MLNC population, we next wondered which population of MLNC is specifically affected by L. brevis supernatants. Thereby, the LC3II expression was monitored after the intracellular staining 37 of CQ treated MLNC, 24 h after the treatment with supernatants. The www.nature.com/scientificreports www.nature.com/scientificreports/ discrimination between T, NKT and NK cells was monitored according to CD3 and CD161 surface staining, as previously described 38 .…”

Section: Supernatants From Lactobacillus Brevis Bgzls10-17 Induce Autmentioning

confidence: 99%

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

Bajić

Đokić

Dinić

et al. 2020

Sci Rep

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The characterization of mechanisms involved in the positive effects of probiotic bacteria in various pathophysiological conditions is a prerogative for their safe and efficient application in biomedicine. We have investigated the immunological effects of live bacteria-free supernatant collected from GABAproducing Lactobacillus brevis BGZLS10-17 on Concanavalin A-stimulated mesenteric lymph node cells (MLNC), an in vitro model of activated immune cells. We have shown that GABA containing and GABA-free supernatant of Lactobacillus brevis BGZLS10-17 have strong immunoregulatory effects on MLNC. Further, GABA produced by this strain exhibit additional inhibitory effects on proliferation, IFN-γ and IL-17 production by MLNC, and the expression of MHCII and CD80 on antigen presenting cells. At the other hand, GABA-containing supernatants displayed the strongest stimulatory effects on the expression of immunoregulatory molecules, such as Foxp3 + , IL-10, TGF-β, CTLA4 and SIRP-α. By looking for the mechanisms of actions, we found that supernatants produced by BGZLS10-17 induce autophagy in different MLNC, such as CD4 + and CD8 + T lymphocytes, NK and NKT cells, as well as antigen presenting cells. Further, we showed that the stimulation of Foxp3 + , IL-10 and TGF-β expression by BGZLS10-17 produced GABA is completely mediated by the induction of ATG5 dependent autophagy, and that other molecules in the supernatants display GABA-, ATG5-, Foxp3 +-, IL-10-and TGF-β-independent, immunoregulatory effects.

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Section: Supernatants From Lactobacillus Brevis Bgzls10-17 Induce Autmentioning

confidence: 99%

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

Bajić

Đokić

Dinić

et al. 2020

Sci Rep

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“…CQ and Tg are drugs known to induce ER stress, cause G 1 cell cycle arrest and initiate autophagy and to induce significant cell death by apoptosis and in this study by necrosis too. Both drugs were shown to cause G 1 cell cycle arrest in a dose dependent manner and the presence of ER stress was confirmed by the novel use of a range of flow cytometric assays developed in this laboratory . First PERK was shown to be up‐regulated in a dose dependent manner in live cells with a build‐up of misfolded proteins within the ER which was inversely proportional to the dose of the drug.…”

Section: Discussionmentioning

confidence: 89%

“…LC3B is known to be up‐regulated in a cell cycle dependent manner during the autophagic process . Hence this study investigated whether the autophagic biological marker, LC3B and markers of ER stress showed a cell cycle dependent regulation when cells undergo simultaneously ER stress, autophagy, cell cycle arrest, and death .…”

Section: Introductionmentioning

confidence: 98%

A Flow Cytometric Study of ER Stress and Autophagy

2018

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The mechanistic link between ER stress, autophagy, and resultant cell death was investigated by the use of drugs Thapsigargin (Tg) and Chloroquine (CQ) with prior induction and or blockade of autophagy and apoptosis which modulated the ER stress response and resultant form of cell death. All these biological processes can be measured flow cytometrically allowing the determination of the type of cell death, G 1 cell cycle arrest, cell cycle dependent measurement of ER stress transducer PERK, misfolded proteins, reticulophagy, and autophagy marker LC3B. Jurkat cells after Tg or CQ treatment became necrotic and apoptotic, showed G 1 cell cycle arrest, autophagy, and ER stress. Prior induction of autophagy before ER stress increased levels of necrotic and apoptotic cell death. Autophagy was further up-regulated, while PERK was reduced or abrogated. CQ showed reduced levels of misfolded proteins and reticulophagy, while Tg showed no change in misfolded protein levels but increased reticulophagy and thus displayed more ER stress. Prior blockade of apoptosis before induction of ER stress resulted in cell survival except with high Tg levels which induced necrosis. Autophagy was up-regulated with modulation of PERK and reticulophagy levels with an abrogation of the misfolded protein response. Blockade of apoptosis with induction of autophagy before ER stress showed death by necrosis with high dose drugs and cell survival with low doses of drugs. CQ induced reduced levels G 1 cell cycle arrest while it was maintained with Tg. Autophagy was also maintained with reduced levels of ER stress. These data demonstrates a profound link between the processes of ER stress, autophagy, and the resultant form of cell death all of which can be modulated depending upon the sequence and concentration of drugs employed.

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“…Median fluorescent intensity (MFI) of LC3-II-AF-647 of the 660/20 nm signal of the cells was compared to determine relative increase in the LC3-II autophagic signal above resting control cells (n = 3). 16 LTG labeling. To determine the autophagic flux, parental and resistant MCF7 cells were loaded LTG at a final concentration of 50 nM and incubated for 1 hour at 37°C.…”

Section: Detection Of Autophagymentioning

confidence: 99%

Contribution of Autophagy in Acquired Drug Resistance of Human Breast Cancer Cells MCF7 to Doxorubicin

Amani

Shaki

Shokrzadeh

2019

Applied In Vitro Toxicology

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Introduction: At present, autophagy has attracted increased attention as a potential therapeutic target for breast cancer. Many investigators are trying to overcome drug resistance by inhibition of autophagy. The development of in vitro drug-resistant cell lines with well-characterized autophagy-related markers is required to discover new autophagy inhibitors. Therefore, in this study we established resistant human breast cancer cells MCF7 to doxorubicin (DOX) and used flow cytometric assays to study autophagic flux. Materials and Methods: Resistant MCF7 subline (MCF7.res) was derived from parental MCF7 cells (MCF7.par) by continuous exposure to stepwise increasing concentrations of DOX (30-54 lM). Autophagic flux was assessed by the antibody labeling of the microtubule-associated protein LC3-II and analyzed by flow cytometry. In addition, lysosomal mass was measured flow cytometrically by LysoTracker Green (LTG) staining. The median fluorescence intensity of anti-LC3-II and LTG was compared between parental and resistant MCF7 cells. For detection of apoptotic cell death, Annexin V/propidium iodide staining was performed. After *6 months, MCF7.res subline was obtained from MCF7.par cells. Results: DOX resistance was confirmed by measurement of fold resistance and growth curve analysis. Our established MCF7.res subline exhibited 7.1-fold resistance to DOX compared with MCF7.par cells. Flow cytometric analysis revealed that LC3-II level and LTG signal were elevated in MCF7.res compared with MCF7.par, elucidating increased autophagic flux. The results of apoptosis assay showed that chloroquine (an autophagy inhibitor) could reverse drug resistance of MCF7.res by inhibiting autophagy. Conclusion: Our established DOX-resistant MCF7 cells model is reliable and applicable for investigating new autophagy inhibitors.

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Measurement of Autophagy by Flow Cytometry

Cited by 16 publications

References 33 publications

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

A Flow Cytometric Study of ER Stress and Autophagy

Contribution of Autophagy in Acquired Drug Resistance of Human Breast Cancer Cells MCF7 to Doxorubicin

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