Cellular Redox Profiling Using High-content Microscopy

Sieprath, Tom; Corne, Tobias; Robijns, Joke; Koopman, Werner J.H.; Vos, Winnok H. De

doi:10.3791/55449

Cited by 4 publications

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References 37 publications

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“…Levels of intracellular ROS and mitochondrial membrane potential were measured as described elsewhere (Sieprath et al, 2016;Sieprath et al, 2017). Briefly, (sham-)irradiated endothelial cells were grown in 96-well plates and stained for 25 min at room temperature with 2 µM chloromethyl-2 ,7dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) (Life Technologies) together with 100 nM tetramethyl rhodamine methyl ester (TMRM, Invitrogen).…”

Section: High Content Live Cell Imaging Of Intracellular Ros and Mitomentioning

confidence: 99%

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction

et al. 2020

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Background and Purpose: Up to 50-60% of all cancer patients receive radiotherapy as part of their treatment strategy. However, the mechanisms accounting for increased vascular risks after irradiation are not completely understood. Mitochondrial dysfunction has been identified as a potential cause of radiation-induced atherosclerosis.Materials and Methods: Assays for apoptosis, cellular metabolism, mitochondrial DNA content, functionality and morphology were used to compare the response of endothelial cells to a single 2 Gy dose of X-rays under basal conditions or after pharmacological treatments that either reduced (EtBr) or increased (rosiglitazone) mitochondrial content.Results: Exposure to ionizing radiation caused a persistent reduction in mitochondrial content of endothelial cells. Pharmacological reduction of mitochondrial DNA content rendered endothelial cells more vulnerable to radiation-induced apoptosis, whereas rosiglitazone treatment increased oxidative metabolism and redox state and decreased the levels of apoptosis after irradiation.Conclusion: Pre-existing mitochondrial damage sensitizes endothelial cells to ionizing radiation-induced mitochondrial dysfunction. Rosiglitazone protects endothelial cells from the detrimental effects of radiation exposure on mitochondrial metabolism and oxidative stress. Thus, our findings indicate that rosiglitazone may have potential value as prophylactic for radiation-induced atherosclerosis.

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Section: High Content Live Cell Imaging Of Intracellular Ros and Mitomentioning

confidence: 99%

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction

et al. 2020

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The gill epithelial cell lines RTgill-W1, from Rainbow trout and ASG-10, from Atlantic salmon, exert different toxicity profiles towards rotenone

et al. 2022

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In order to ensure the proper use and interpretation of results from laboratory test systems, it is important to know the characteristics of your test system. Here we compare mitochondria and the handling of reactive oxygen species (ROS) in two gill epithelial cell lines, the well-known RTgill-W1 cell line from Rainbow trout and the newly established ASG-10 cell line from Atlantic salmon. Rotenone was used to trigger ROS production. Rotenone reduced metabolic activity and induced cell death in both cell lines, with RTgill-W1 far more sensitive than ASG-10. In untreated cells, the mitochondria appear to be more fragmented in RTgill-W1 cells compared to ASG-10 cells. Furthermore, rotenone induced mitochondrial fragmentation, reduced mitochondria membrane potential (Δψm) and increased ROS generation in both cell lines. Glutathione (GSH) and catalase is important to maintain the cellular oxidative balance by eliminating hydrogen peroxide (H2O2). In response to rotenone, both GSH and catalase depletion were observed in the RTgill-W1 cells. In contrast, no changes were found in the GSH levels in ASG-10, while the catalase activity was increased. In summary, the two salmonid gill cell lines have different tolerance towards ROS, probably caused by differences in mitochondrial status as well as in GSH and catalase activities. This should be taken into consideration with the selection of experimental model and interpretation of results. Graphical abstract

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Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse

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et al. 2020

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26The detection and neutralization of infected cells and tumors by cytotoxic lymphocytes is a 27 vital immune defense mechanism. The immunological synapse orchestrates the target 28 recognition process and the subsequent cytotoxic activity. Here, we present an integrated 29 experimental and computational strategy to systematically characterize the morphological 30properties of the immunological synapse of human cytotoxic lymphocytes. Our approach 31 combines high-content imaging with an unbiased, data-driven identification of high-32 resolution morphological profiles. Such profiling discriminates with high accuracy 33 immunological synapse perturbations induced by an array of actin drugs in both model cell 34 lines and primary lymphocytes. It reveals inter-individual heterogeneity in lymphocyte 35 morphological traits. Furthermore, it uncovers immunological synapse alterations in 36 functionally defective CD8 + T cells from immunodeficient patients carrying ARPC1B 37 mutations. Our study thus provides a foundation for the application of morphological 38 profiling as a powerful and scalable approach to monitor lymphocyte activation status in 39 experimental and disease settings. 40 41 42The immunological synapse (IS) is a complex cellular structure that sets lymphocyte 43 activation and function during encounter with antigen-presenting cells and target cells. The 44 canonical IS is characterized by a symmetrical architecture consisting of concentric rings of 45 F-actin and integrins, while the antigen receptors occupy a central position 1,2 . The 46 lymphocyte spreading associated with IS assembly, as well as the molecular organization 47 defining IS architecture, rely on actin cytoskeleton dynamics. In cytotoxic lymphocytes, 48 including CD8+ T cells and NK cells, the IS is particularly important because it sustains the 49 polarized delivery of cytolytic molecules such as perforin and granzymes towards target 50 cells 3 . Indeed, activation of the integrin LFA-1 via an inside-out signaling from the T-cell 51 receptor (TCR) in T cells, and several stimulatory receptors in NK cells 4-7 , leads to the 52 formation of a tight adhesive ring allowing confinement of the degranulation process. 53Additional layers of control of lytic granule delivery at the IS are their polarization via the 54 orientation of the microtubule organizing center 8,9 and their restricted passage through 55 pervasive actin cytoskeleton clearances 10 . Given the key events occurring at the IS, this 56 structure is a window of choice to monitor lymphocyte activation and function. Indeed, the 57 positioning and dynamic behavior of multiple receptors and signaling molecules have been 58 characterized within the IS 11 , and alterations of the its architecture have been reported in 59 multiple disease settings 12,13 . However, the various microscopy approaches employed so far 60 to characterize spatial organization of the IS have remained low throughput and have been 61 restricted to the analysis of a limited number of morphological features. A more ...

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Cellular Redox Profiling Using High-content Microscopy

Cited by 4 publications

References 37 publications

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction

Rosiglitazone Protects Endothelial Cells From Irradiation-Induced Mitochondrial Dysfunction

The gill epithelial cell lines RTgill-W1, from Rainbow trout and ASG-10, from Atlantic salmon, exert different toxicity profiles towards rotenone

Morphological profiling of human T and NK lymphocytes identifies actin-mediated control of the immunological synapse

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