Functional Heterogeneity of an Isolated Mitochondrial Population Revealed by Cytofluorometric Analysis at the Single Organelle Level

Cossarizza, Andrea; Ceccarelli, Daniela; Masini, Alberto

doi:10.1006/excr.1996.0011

Cited by 205 publications

(144 citation statements)

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“…In a previous study we demonstrated a good reproducibility of the assay used [27]. JC-1 has no concentration-dependent direct effect on DC m [29]; it produces reliable results for analysing DC m in contrast to other dyes (CMXRos, Rho123, DiOC6) because of its high sensitivity to changes of plasma membrane potential or nonspecific binding [30,31]. Cell staining was performed as follows: PBMC cell suspension was adjusted to a concentration of 1.0 Â 10 6 cells/mL and incubated in complete medium (RPMI 1640 with L-glutamine, 5% foetal calf serum, 1% penicillin/streptomycin) (PAA Laboratories GmbH, Pasching, Austria) with JC-1 (10 mg/mL) for 15 min at 37 1C and 5% CO 2 in the dark [14,31].…”

Section: Patient Specimen Collectionmentioning

confidence: 82%

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

et al. 2009

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ObjectivesInfection with HIV leads to progressive CD4 T-cell loss, resulting in AIDS. Apoptosis is the main mechanism for the loss of infected and bystander cells, but the complex interacting factors inducing and inhibiting apoptosis are not fully understood. Mitochondrial dysfunction is a pivotal step of the apoptotic cascade and can result in reduced mitochondrial membrane potential. MethodsThe mitochondrial membrane potential of peripheral blood mononuclear cells (PBMC) was measured by flow cytometry using the dye JC-1 (Molecular Probes Inc). Apoptotic cells were identified using the Annexin V assay (Becton Dickinson GmbH). ResultsThe mitochondrial membrane potential of PBMC was significantly decreased and apoptotic cell rate was increased in HIV-infected therapy-naïve patients compared with HIV-negative controls. There was a highly significant correlation between the mitochondrial membrane potential and the rate of apoptosis. CD4 cell count was correlated negatively to the apoptotic rate and positively to the mitochondrial membrane potential. ConclusionsThe JC-1 assay is a sensitive tool to detect changes of mitochondrial membrane potential associated with apoptosis in HIV-infected therapy-naïve patients. We could show in vivo that a reduction of mitochondrial membrane potential is correlated to apoptosis of PBMC, CD4 cell count and HIV viral load during HIV infection. IntroductionInfection with HIV leads to progressive CD4 T-cell death, resulting in the development of AIDS. The mechanisms triggering this CD4 T-cell death are still not understood fully, but data indicate that apoptosis plays a major role [1]. There is a correlation between the extent of apoptosis and disease progression [2,3], and highly active antiretroviral therapy is associated with a lower level of CD4 T-cell apoptosis in HIV-1 infected patients [4][5][6][7].Both infected and uninfected CD4 T-cells can die during HIV-1 infection by different cell death pathways, but HIV-1 induced bystander CD4 T-cell demise is now recognized as pivotal to the development of immunodeficiency. Chronically increased activation of the immune system may contribute directly to progressive CD4 T-cell depletion, irrespective of viral load [8,9]. Upon receiving a death signal, a cascade of molecular events resulting in the activation or inactivation of numerous cellular proteins is initiated, leading to morphological and biochemical changes such as plasma membrane blebbing, DNA fragmentation and mitochondrial dysfunction. The regulators of the apoptotic pathway exert their function primarily at the mitochondrion by either preventing or inducing mitochondrial dysfunction [10,11]. Furthermore, there is evidence that resistance to apoptosis in persistently infected cells involves direct modulation of the mitochondrial pathway [1]. The measurement of the mitochondrial membrane potential of peripheral blood mononuclear cells (PBMC) might be a useful tool to [24]. In the early stages of apoptosis, there are changes at the cell surface. One of these plasma membrane al...

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Section: Patient Specimen Collectionmentioning

confidence: 82%

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

et al. 2009

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“…Dissipation of ⌬⌿ m can be judged by flow cytometry measuring a decrease of FL-2 which correlates with an impaired formation of JC-1 aggregates. 42 As shown in Figure 4b …”

Section: Ajoene Induces Mitochondrial Membrane Permeabilization (Mpp)mentioning

confidence: 82%

Ajoene, an experimental anti-leukemic drug: mechanism of cell death

et al. 2002

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The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoenetriggered apoptosis. We show here that ajoene (20 M) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x L clearly diminished ajoeneinduced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8. Leukemia (2002) 16, 74-83.

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“…The JC-1 aggregate/monomer ratio is assumed to be proportional to mitochondrial membrane potential (Reers et al, 1991;Cossarizza et al, 1996). FL1 and FL2 fluorescence intensities are volume-dependent when their ratio is constant at a given membrane potential (Reers et al, 1991).…”

Section: Assessment Of Mitochondrial Membrane Potentialmentioning

confidence: 99%

Effects of Alexandrium minutum exposure upon physiological and hematological variables of diploid and triploid oysters, Crassostrea gigas

et al. 2010

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minutum compared to control oysters. At the same time, many oysters exposed to the harmful microalgae presented a strong inflammatory response in different tissues of the digestive gland: in intestine as well as in digestive ducts and tubules. Spermatozoa in oysters exposed to A. minutum were morphologically and functionally modified compared to spermatozoa of control oysters. Indeed, spermatozoa were less motile and had lower ATP content in oysters exposed to A. minutum. Meanwhile, spermatozoa produced by control oysters showed higher 2 percentage of mortality and relative DNA content than those produced by A. minutum exposed oysters. Finally, the characteristics of the mitochondria of spermatozoa also appeared to be modified upon A. minutum exposure. The results of this study suggests that an exposure of oysters to A. minutum, reducing energy status and motility of spermatozoa associated to morphological changes at the cellular and sub-cellular levels, can have consequences on spermatozoa fertility and reproduction success.

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Functional Heterogeneity of an Isolated Mitochondrial Population Revealed by Cytofluorometric Analysis at the Single Organelle Level

Cited by 205 publications

References 0 publications

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

Ajoene, an experimental anti-leukemic drug: mechanism of cell death

Effects of Alexandrium minutum exposure upon physiological and hematological variables of diploid and triploid oysters, Crassostrea gigas

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