Measuring apoptosis by microscopy and flow cytometry

Henry, Conor M.; Hollville, Émilie; Martin, Séamus J.

doi:10.1016/j.ymeth.2013.01.008

Cited by 149 publications

(89 citation statements)

References 32 publications

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“…Of note, apoptosis and necroptosis are morphologically distinct. Apoptotic cells are characterized by phosphatidylserine exposure on the cell surface followed by membrane blebbing, nuclear fragmentation, decreased cellular volume and formation of apoptotic bodies56. In contrast, necroptotic cells include early plasma membrane rupture followed by rapid cytoplasmic and nuclear swelling and organelle breakdown38.…”

Section: Discussionmentioning

confidence: 99%

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection

Just

Nishanth

Buchbinder

et al. 2016

Sci Rep

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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.

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Section: Discussionmentioning

confidence: 99%

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection

Just

Nishanth

Buchbinder

et al. 2016

Sci Rep

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“…DNA laddering during apoptosis was characterized by agarose gel electrophoresis 40 . In brief, 60,000 Jurkat cells were induced to undergo apoptosis by anti-Fas treatment for 2h and lysed in TES lysis buffer containing RNase for 2h at 37°C.…”

Section: Online-only Methodsmentioning

confidence: 99%

Unexpected link between an antibiotic, pannexin channels and apoptosis

Poon

Chiu

Armstrong

et al. 2014

Nature

216

274

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Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. In a small molecule screen, we discovered the quinolone antibiotic trovafloxacin as a novel PANX1 inhibitor. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels, and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials.

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“…The sample was additionally visualized under a confocal microscope (OLYMPUS FV1000, Japan). The Annexin V+/PI- osteoblasts were considered early apoptotic cells (Henry, Hollville & Martin, 2013). The Annexin V+/PI+ osteoblasts were considered late apoptotic cells (Henry, Hollville & Martin, 2013).…”

Section: Methodsmentioning

confidence: 99%

Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway

Tian

Dai

et al. 2016

PeerJ

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BackgroundIron overload is recognized as a new pathogenfor osteoporosis. Various studies demonstrated that iron overload could induce apoptosis in osteoblasts and osteoporosis in vivo. However, the exact molecular mechanisms involved in the iron overload-mediated induction of apoptosis in osteoblasts has not been explored.PurposeIn this study, we attempted to determine whether the mitochondrial apoptotic pathway is involved in iron-induced osteoblastic cell death and to investigate the beneficial effect of N-acetyl-cysteine (NAC) in iron-induced cytotoxicity.MethodsThe MC3T3-E1 osteoblastic cell line was treated with various concentrations of ferric ion in the absence or presence of NAC, and intracellular iron, cell viability, reactive oxygen species, functionand morphology changes of mitochondria and mitochondrial apoptosis related key indicators were detected by commercial kits. In addition, to further explain potential mechanisms underlying iron overload-related osteoporosis, we also assessed cell viability, apoptosis, and osteogenic differentiation potential in bone marrow-derived mesenchymal stemcells(MSCs) by commercial kits.ResultsFerric ion demonstrated concentration-dependent cytotoxic effects on osteoblasts. After incubation with iron, an elevation of intracelluar labile iron levels and a concomitant over-generation of reactive oxygen species (ROS) were detected by flow cytometry in osteoblasts. Nox4 (NADPH oxidase 4), an important ROS producer, was also evaluated by western blot. Apoptosis, which was evaluated by Annexin V/propidium iodide staining, Hoechst 33258 staining, and the activation of caspase-3, was detected after exposure to iron. Iron contributed to the permeabilizatio of mitochondria, leading to the release of cytochrome C (cyto C), which, in turn, induced mitochondrial apoptosis in osteoblasts via activation of Caspase-3, up-regulation of Bax, and down-regulation of Bcl-2. NAC could reverse iron-mediated mitochondrial dysfunction and blocked the apoptotic events through inhibit the generation of ROS. In addition, iron could significantly promote apoptosis and suppress osteogenic differentiation and mineralization in bone marrow-derived MSCs.ConclusionsThese findings firstly demonstrate that the mitochondrial apoptotic pathway involved in iron-induced osteoblast apoptosis. NAC could relieved the oxidative stress and shielded osteoblasts from apoptosis casused by iron-overload. We also reveal that iron overload in bone marrow-derived MSCs results in increased apoptosis and the impairment of osteogenesis and mineralization.

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Measuring apoptosis by microscopy and flow cytometry

Cited by 149 publications

References 32 publications

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection

A20 Curtails Primary but Augments Secondary CD8+ T Cell Responses in Intracellular Bacterial Infection

Unexpected link between an antibiotic, pannexin channels and apoptosis

Iron overload induced death of osteoblasts in vitro: involvement of the mitochondrial apoptotic pathway

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