Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

2007 ◽  
Vol 2 (11) ◽  
pp. 2719-2727 ◽  
Author(s):  
Leonarda Troiano ◽  
Roberta Ferraresi ◽  
Enrico Lugli ◽  
Elisa Nemes ◽  
Erika Roat ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Multiparametric Analysis ◽  
Polychromatic Flow Cytometry

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 UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

2021 ◽  
Author(s):  
ChaoYong Liu ◽  
YanMin Ma ◽  
XiaoQin Zhang ◽  
Yang Liu ◽  
XiaoCheng Yin
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Chronic Myeloid Leukaemia ◽  
Western Blot ◽  
Myeloid Leukaemia ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
K562 Cells ◽  
Mitochondrial Mass ◽  
Related Proteins

Abstract Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.

Sign in / Sign up

Export Citation Format

Share Document