scholarly journals A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry

Cytometry ◽  
1994 ◽  
Vol 15 (4) ◽  
pp. 335-342 ◽  
Author(s):  
Gloria Juan ◽  
Marika Cavazzoni ◽  
Guillermo T. Sáez ◽  
José-Enrique O'Connor
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Kinetic Method ◽  
Isolated Hepatocytes ◽  
Rhodamine 123 ◽  
Fast Kinetic

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

The Inhibitor of Cyclin-Dependent Kinase4a (INK4a) Signaling Pathway Induces Aging in Human Skeletal Muscle Myoblasts and Decreases the Mitochondrial Membrane Potential

2019 ◽  
Vol 9 (9) ◽  
pp. 1192-1198
Author(s):  
Dong Yan ◽  
Xiang Liao ◽  
Li-Xia Zhao ◽  
Chang-Hong Xiao
Keyword(s):  
Skeletal Muscle ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Signaling Pathway ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Human Skeletal Muscle ◽  
Lentiviral Vector ◽  
Accelerated Aging ◽  
Senescence Phenotype

The effect of the inhibitor of cyclin-dependent kinase4a (INK4a) signaling pathway on myoblastic aging was studied in this paper. Human skeletal muscle myoblasts were transfected with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and RT-qPCR and western blotting were used to identify p16INK4a gene transcription and protein expression. The degree of cell senescence was assessed using Senescence-associated β-galactosidase staining. flow cytometry and JC-1 staining was used to analyze the mitochondrial membrane potential (MMP). The senescence phenotype was observed in myoblasts transfected with p16INK4a, the MMP was significantly decrease in p16INK4a-transfected myoblasts, while the MMP was decreased only slightly in control cells. Upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activated the mitochondrial pro-aging pathway by reducing the MMP, which indirectly accelerated aging in myoblasts.

scholarly journals Neuroprotective Effects of Idebenone on hydrogen peroxide (H2O2)-induced Oxidative Damage in Retinal ganglion cell-5 (RGC-5) Cells

2019 ◽  
Author(s):  
Yuping Wang ◽  
Jing Wang ◽  
Xi Zhang ◽  
Yifan Feng ◽  
Yuanzhi Yuan
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Oxidative Damage ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Beclin 1 ◽  
Control Group ◽  
Related Protein ◽  
H2o2 Treatment ◽  
Cyt C

Abstract Purposes To investigated the neuroprotective effect of Idebenone against H2O2-induced oxidative damage in RGC-5 cells. Methods RGC-5 cells were treated with different concentrations (5, 10, 20μM) of idebenone for 12h prior to addition of 300µM H2O2 for 12 h. The apoptosis of RGC-5 cells were detected by flow cytometry. The changes of mitochondrial membrane potential were detected by JC-1 staining. The autophagy in RGC-5 cells was observed by transmission electron microscopy, and the expression level of autophagy-related protein light chain3, Beclin-1 and mitochondrial membrane potential-related protein Cyt-c in RGC-5 cells were measured by Western blot analysis. Results Flow cytometry showed that the apoptosis rates in control group, H2O2 group and H2O2-treatment with Idebenone pretreatment groups were (6.48±0.55)%, (27.34±0.51)%, (22.88±0.52)%, (15.45±0.81)%, (12.59±0.58)%, respectively(F = 559.7, P <0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, while which was decreased in H2O2-treatment with Idebenone pretreatment groups. After incubation of RGC-5 cells with H2O2, the mitochondrial membrane potential was significantly decreased, while idebenone could prevent the decrease of MMP. Contrast with control group, LC3 II /I, the expression levels of Beclin-1 and Cyt-c in H2O2 group increased significantly(P<0.05); while contrast with H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c in H2O2-treatment with Idebenone pretreatment groups was significantly decreased(P<0.05). Conclusion Idebenone may have protective effects on RGC-5 cells suffering from oxidative damage induced by H2O2 through improving antioxidant capacity, reducing mitochondrial membrane potential decline and the activity of autophagy.

scholarly journals Rise in cytosolic Ca2+ and collapse of mitochondrial potential in anoxic, but not hypoxic, rat proximal tubules.

1996 ◽  
Vol 7 (11) ◽  
pp. 2348-2356
Author(s):  
S M Peters ◽  
M J Tijsen ◽  
R J Bindels ◽  
C H Van Os ◽  
J F Wetzels
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Cell Injury ◽  
Cell Damage ◽  
Energy State ◽  
Oxygen Deprivation ◽  
Proximal Tubules ◽  
Rhodamine 123 ◽  
Anoxic Conditions

It has been suggested that ischemic renal proximal tubular cell injury is mediated by an increase in cytosolic calcium concentrations ((Ca2+)i). However, measurements of (Ca2+)i in rat or rabbit proximal tubules exposed to hypoxia or anoxia have yielded ambiguous results. This study explored the possibility that the severity of oxygen deprivation and the energy state of the mitochondria are important determinants of (Ca2+)i. To this end, (Ca2+)i (measured with fura-2) and the mitochondrial membrane potential (measured with rhodamine 123) were studied simultaneously in individual rat proximal tubules in hypoxic and anoxic conditions. (Ca2+)i did not change during hypoxia, but increased rapidly during anoxia. Increases in (Ca2+)i were only observed in parallel with a decrease of rhodamine 123 fluorescence, which indicates a collapse of the mitochondrial membrane potential. The increase in (Ca2+)i during anoxia was prevented by incubating the tubules in a low Ca2+ medium, which did not interfere with the collapse of the mitochondrial membrane potential. Both hypoxic and anoxic incubation led to cell death, as assessed by the fluorescent dye propidium iodide. These results clearly demonstrate that the level of oxygen deprivation is critical in determining changes in (Ca2+)i. Because cell damage occurred in both hypoxic and anoxic conditions. It was concluded that an increase in (Ca2+)i is not a necessary prerequisite for the development of ischemic cell injury.

Mitochondrial membrane potential regulation is independent of c-fos expression

1999 ◽  
Vol 77 (3) ◽  
pp. 195-203
Author(s):  
Roger A Moorehead ◽  
Gurmit Singh
Keyword(s):  
Membrane Potential ◽  
Ovarian Carcinoma ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Potentials ◽  
Rhodamine 123 ◽  
Parental Line ◽  
Rat Fibroblasts ◽  
Human Ovarian Carcinoma ◽  
Fos Expression

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.Key words: c-fos, mitochondria, membrane potential, rhodamine 123 (Rh123), lipophilic cations.

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