Quantification of apoptosis and necroptosis at the single cell level by a combination of Imaging Flow Cytometry with classical Annexin V/propidium iodide staining
“…There are major differences in the morphology of the apoptotic vs. necroptotic cells that allow to distinguish between these two types of cell death. In particular, the necroptotic cells are typically swollen, while apoptotic cells undergo shrinkage during the course of cell death38. The identification of Lm OVA-specific CD8 + T cells undergoing apoptosis or necroptosis was performed by imaging flow cytometry (IFC), allowing the simultaneous analysis of a single cell by flow cytometry and microscopy to differentiate apoptosis from necroptosis38.…”
The ubiquitin-modifying enzyme A20, an important negative feedback regulator of NF-κB, impairs the expansion of tumor-specific CD8+ T cells but augments the proliferation of autoimmune CD4+ T cells. To study the T cell-specific function of A20 in bacterial infection, we infected T cell-specific A20 knockout (CD4-Cre A20fl/fl) and control mice with Listeria monocytogenes. A20-deficient pathogen-specific CD8+ T cells expanded stronger resulting in improved pathogen control at day 7 p.i. Imaging flow cytometry revealed that A20-deficient Listeria-specific CD8+ T cells underwent increased apoptosis and necroptosis resulting in reduced numbers of memory CD8+ T cells. In contrast, the primary CD4+ T cell response was A20-independent. Upon secondary infection, the increase and function of pathogen-specific CD8+ T cells, as well as pathogen control were significantly impaired in CD4-Cre A20fl/fl mice. In vitro, apoptosis and necroptosis of Listeria-specific A20-deficient CD8+ T cells were strongly induced as demonstrated by increased caspase-3/7 activity, RIPK1/RIPK3 complex formation and more morphologically apoptotic and necroptotic CD8+ T cells. In vitro, A20 limited CD95L and TNF-induced caspase3/7 activation. In conclusion, T cell-specific A20 limited the expansion but reduced apoptosis and necroptosis of Listeria-specific CD8+ T cells, resulting in an impaired pathogen control in primary but improved clearance in secondary infection.
“…There are major differences in the morphology of the apoptotic vs. necroptotic cells that allow to distinguish between these two types of cell death. In particular, the necroptotic cells are typically swollen, while apoptotic cells undergo shrinkage during the course of cell death38. The identification of Lm OVA-specific CD8 + T cells undergoing apoptosis or necroptosis was performed by imaging flow cytometry (IFC), allowing the simultaneous analysis of a single cell by flow cytometry and microscopy to differentiate apoptosis from necroptosis38.…”
The ubiquitin-modifying enzyme A20, an important negative feedback regulator of NF-κB, impairs the expansion of tumor-specific CD8+ T cells but augments the proliferation of autoimmune CD4+ T cells. To study the T cell-specific function of A20 in bacterial infection, we infected T cell-specific A20 knockout (CD4-Cre A20fl/fl) and control mice with Listeria monocytogenes. A20-deficient pathogen-specific CD8+ T cells expanded stronger resulting in improved pathogen control at day 7 p.i. Imaging flow cytometry revealed that A20-deficient Listeria-specific CD8+ T cells underwent increased apoptosis and necroptosis resulting in reduced numbers of memory CD8+ T cells. In contrast, the primary CD4+ T cell response was A20-independent. Upon secondary infection, the increase and function of pathogen-specific CD8+ T cells, as well as pathogen control were significantly impaired in CD4-Cre A20fl/fl mice. In vitro, apoptosis and necroptosis of Listeria-specific A20-deficient CD8+ T cells were strongly induced as demonstrated by increased caspase-3/7 activity, RIPK1/RIPK3 complex formation and more morphologically apoptotic and necroptotic CD8+ T cells. In vitro, A20 limited CD95L and TNF-induced caspase3/7 activation. In conclusion, T cell-specific A20 limited the expansion but reduced apoptosis and necroptosis of Listeria-specific CD8+ T cells, resulting in an impaired pathogen control in primary but improved clearance in secondary infection.
“…Martin et al [66] found that the presence of phosphatidylserine in cell death assays with annexin V-FITC is considered an early event of apoptosis. Cell labeling with propidium iodide, which binds to DNA, is only observed during membrane damage, which occurs in late apoptosis or early necroptosis [67]. The flavonoids that are catechin and quercetin derivatives have potent cytotoxic activities and cause apoptosis using mitochondrial pathways in human leukemic cell lines, including monocytic leukemia (U937), erythroleukemia (K562), and promyelocytic leukemia (HL-60) [25].…”
Hancornia speciosa Gomes (Apocynaceae) is a fruit tree, popularly known as mangabeira, and it is widely distributed throughout Brazil. Several parts of the plant are used in folk medicine, and the leaf and bark extracts have anti-inflammatory, antihypertensive, antidiabetic, and antimicrobial properties. In this study, we investigated the chemical composition of the ethanolic extract of Hancornia speciosa leaves (EEHS) and its antioxidant, antimicrobial, and cytotoxic activities as well as the mechanisms involved in cell death. The chemical compounds were identified by liquid chromatography coupled to mass spectrometry (LC-MS/MS). The antioxidant activity of the EEHS was investigated using the method that involves the scavenging of 2,2-diphenyl-1-picrylhydrazyl free radicals as well as the inhibition of oxidative hemolysis and lipid peroxidation induced by 2,2’-azobis (2-amidinopropane) in human erythrocytes. The antimicrobial activity was determined by calculating the minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration, and zone of inhibition. Kasumi-1 leukemic cells were used to assess the cytotoxic activity and mechanisms involved in cell death promoted by the EEHS. The chemical compounds identified were quinic acid, chlorogenic acid, catechin, rutin, isoquercitrin, kaempferol-rutinoside, and catechin-pentoside. The EEHS demonstrated antioxidant activity via the sequestration of free radicals, inhibition of hemolysis, and inhibition of lipid peroxidation in human erythrocytes incubated with an oxidizing agent. The antimicrobial activity was observed against American Type Culture Collection (ATCC) and hospital strains of bacteria and fungi, filamentous fungi and dermatophytes. The cytotoxic activity of the EEHS was induced by apoptosis, reduction of the mitochondrial membrane potential, and activation of cathepsins. Together, these results indicate the presence of phenolic compounds and flavonoids in the EEHS and that their antioxidant, antimicrobial, and cytotoxic activities in acute myeloid leukemia cells are mediated by apoptosis.
“…Fluorescently conjugated annexin A5 is a well-established tool for the detection of apoptotic cells in vitro (i.e. FITC-conjugated Annexin V [137]). More recently, Technetium-99 m (99 m Tc) radiolabelled annexin A5 has been used in vivo to detect apoptosis in over 30 clinical trials for a range of disorders, including acute cardiac allograft rejection, acute myocardial infarction, ischemic brain injury, cerebral hypoxia, breast cancer, lung cancer, lymphoma, sarcoma, and hepatitis [148].…”
Section: Techniques For Monitoring Retinal Ganglion Cell Healthmentioning
confidence: 99%
“…More recently, Technetium-99 m (99 m Tc) radiolabelled annexin A5 has been used in vivo to detect apoptosis in over 30 clinical trials for a range of disorders, including acute cardiac allograft rejection, acute myocardial infarction, ischemic brain injury, cerebral hypoxia, breast cancer, lung cancer, lymphoma, sarcoma, and hepatitis [148]. Externalisation of phosphatidylserine is also reported in cells undergoing necroptosis, which can also be identified using fluorescently conjugated annexin A5 [137]. Fig.…”
Section: Techniques For Monitoring Retinal Ganglion Cell Healthmentioning
Over 60 million people worldwide are diagnosed with glaucomatous optic neuropathy, which is estimated to be responsible for 8.4 million cases of irreversible blindness globally. Glaucoma is associated with characteristic damage to the optic nerve and patterns of visual field loss which principally involves the loss of retinal ganglion cells (RGCs). At present, intraocular pressure (IOP) presents the only modifiable risk factor for glaucoma, although RGC and vision loss can continue in patients despite well-controlled IOP. This, coupled with the present inability to diagnose glaucoma until relatively late in the disease process, has led to intense investigations towards the development of novel techniques for the early diagnosis of disease. This review outlines our current understanding of the potential mechanisms underlying RGC and axonal loss in glaucoma. Similarities between glaucoma and other neurodegenerative diseases of the central nervous system are drawn before an overview of recent developments in techniques for monitoring RGC health is provided, including recent progress towards the development of RGC specific contrast agents. The review concludes by discussing techniques to assess glaucomatous changes in the brain using MRI and the clinical relevance of glaucomatous-associated changes in the visual centres of the brain.
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