Postnatal changes in rhodamine‐123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by <scp>atp</scp>

Almeida, Ángeles; López-Mediavilla, Casilda; Órfão, Alberto; Medina, José M.

doi:10.1016/0014-5793(94)00345-9

Cited by 13 publications

(13 citation statements)

References 22 publications

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“…Mitochondrial subpopulations have previously been demonstrated using FCM (12,13), and mitochondria within a cell may have a heterogeneous response to deathinducing stimuli (39). However, although variation was relatively large, no mitochondrial subpopulations were found in the present study.…”

Section: Mitochondrial Subpopulationscontrasting

confidence: 54%

“…Flow cytometric analysis of isolated liver mitochondria has been described previously by several investigators. Membrane potential was measured using Rhodamine-123 (Rh123) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), 3,3Ј-dihexyloxacarbocyanine iodide (DiOC 6 (3)) (18,19), or 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) (20). Reactive oxygen species (ROS) generation in isolated brain and liver mitochondria has been measured by flow cytometry using 2Ј,7Ј-dichlorodihydrofluorescein-diacetate (H 2 DCF-DA) (21)(22)(23) or dihydrorhodamine-123 (22,24).…”

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confidence: 99%

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Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death

Mattiasson

2004

Cytometry Pt A

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Background: Mitochondria are key players in many forms of cell death, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes, and release of apoptogenic proteins are involved in these processes. Flow cytometric analysis of isolated mitochondria enables parallel analysis of mitochondrial structure and function in individual mitochondria, and small mitochondrial samples are sufficient for analysis. This article describes a well-characterized protocol for flow cytometric analysis of isolated liver mitochondria that can be used to detect mitochondrial alterations relevant to cell death. Methods: Fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), and to measure membrane potential (tetramethylrhodamine-methylester, 1,1Ј,3,3,3Ј,3Ј-hexamethylindodicarbocyanine-iodide), as well as production of ROS (2Ј,7Ј-dichlorodihydrofluorescein-diacetate). Calcium-induced mitochondrial swelling was detected as a decrease in SSC. To ensure optimal concentrations of all probes, the effect on mitochondrial respiration was evaluated. Results: This protocol can be used to determine the purity of the mitochondrial preparation, to detect calcium-induced morphological changes, small mitochondrial de-and hyperpolarizations, as well as physiological changes in ROS generation. Key terms: mitochondria; cell death; flow cytometry; reactive oxygen species; membrane potentials; mitochondrial permeability transition pore Mitochondrial dysfunction has been implicated as one of the key components in induction of cell death in a number of disease models. Numerous mechanisms have been proposed; for example, increased oxidative stress, altered calcium homeostasis, impairment of respiratory chain complexes, loss of membrane potential, and more recently, the activation of a pore in the inner mitochondrial membrane, the mitochondrial permeability transition pore (mPTP), with release of apoptogenic factors (1-5). Several or all of these changes may be activated in parallel during induction of cell death, posing an analytical challenge. However, using a combination of fluorescent markers and flow cytometry, several of these parameters can be studied simultaneously at the single organelle level. This approach has several advantages: (a) very small samples can be analyzed (5,000 -10,000 mitochondria), (b) preparations contaminated with other cellular constituents can be handled (using mitochondria-specific markers), (c) analysis of covariance of relevant parameters is possible, (d) potential identification of mitochondrial subpopulations, and (e) mitochondria can be sorted for further analysis of, e.g., protein profile. Also, the effects of pharmacologic agents or recombinant proteins of interest are more easily studied in isolated mitochondria than in intact cells. However, fluorescent probes can affect mitochondrial function, which may preclude or seriously limit their use (6), suggesting that a careful characterization of such a flow cytometric protocol has to be under...

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Section: Mitochondrial Subpopulationscontrasting

confidence: 54%

mentioning

confidence: 99%

Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death

Mattiasson

2004

Cytometry Pt A

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“…The enrichment in adenine nucleotides promotes structural changes in the inner mitochondria membrane that result in a rapid increase in their osmotic activity and the enhancement of the ‘de novo’ protein synthesis involved in mitochondrial function [9,16], in particular the catalytic β‐subunit of H + ‐ATP synthase whose synthesis is controlled at the post‐transcriptional level by increasing stability [15] and translational efficiency [26] of mitochondrial transcripts.…”

Section: Changes In Mitochondrial Populations During Perinatal Develomentioning

confidence: 99%

“…The postnatal increase in the LFP is prevented by inhibitors of nuclear DNA (nDNA)‐encoded protein synthesis and to a lesser extent by those of mitochondrial protein synthesis, suggesting that the transition of HFP to LFP depends on protein synthesis [16]. Since hypothyroidism inhibits the transition of HFP to LFP together with the synthesis of β‐subunit of H + ‐ATP synthase, it may be speculated that the transition of HFP to LFP may involve the synthesis of β‐subunit.…”

Section: Changes In Mitochondrial Populations During Perinatal Develomentioning

confidence: 99%

“…Human ¢broblast Sorting of mitochondria for analysis of mDNA 10 N-nonyl-acridine orange [76] WGA: Wheat germ-agglutinin. Con-A: Concanavalin A. DiOC6 (3): dihexlocarbocyanine iodide drial membranes to osmotic pressure observed during perinatal development are mimicked`in vitro' by ATP [16,17], it is reasonable to suggest that adenine nucleotide accumulation is essential for the postnatal assembling of mitochondrial membranes and for the subsequent burst of mitochondrial function (Fig. 2).…”

Section: Analysis Of Ca 2 Mobilization In Mitochondriamentioning

confidence: 99%

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Flow cytometry of isolated mitochondria during development and under some pathological conditions

2001

Self Cite

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Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed. ß

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Impaired development of mitochondria plays a role in the central nervous system defects of fetal alcohol syndrome

Liu

2005

Birth Defects Research

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Postnatal changes in rhodamine‐123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by atp

Cited by 13 publications

References 22 publications

Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death

Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death

Flow cytometry of isolated mitochondria during development and under some pathological conditions

Impaired development of mitochondria plays a role in the central nervous system defects of fetal alcohol syndrome

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