mitochondrial membrane potential assay flow cytometry

The Flow Cytometry section specializes in providing immunophenotyping support of NCI intramural clinical [learn more] Lab Head: Jon Inglefield. Inflammatory markers: Cox-2, TNF-a, IL-6, etc. The control count represents the total possible number of migrated cells. Analytical Flow Cytometry Utilizes a Becton Dickinson FACSCalibur, a 4-color, dual-laser benchtop flow cytometer. 6) were seeded in tissue culture plates and were treated with 18, 27, 36, and 45 g/ml of chalepin for 48 and 72 h incubation. Cells were washed once in PBS, detached, and centrifuged for 5 min at 250 g. Unidentified soluble factors secreted by E. coli, a frequently isolated microorganism in genitourinary infections, have been reported to inhibit mitochondrial membrane potential (m), motility and vitality of human spermatozoa. Website CRE x. A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. Email: jon.inglefield@nih.gov. Mitochondrial uptake of JC-1 is dependent on its lipophilic action driven by polarity of . We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. Mitochondrial electron transport chain complexes activities and mitochondrial membrane potential in sperm increased linearly with dietary ASTA supplementation . Article Snippet: Mitochondrial membrane potential and mtDNA/nDNA measurement Zebrafish cells or human cells were stained with tetramethylrhodamine, methyl ester (Image-iT TMRM Reagent, Invitrogen, ref. At the end of . Flow . Flow cytometric enumeration of (left panel) cells in control wells (no HUVECs) and (right panel) cells that have migrated through wells containing HUVECs. Apoptotic markers: Cas-3, Bcl-2, etc. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. the dye accumulates primarily in mitochondria with active mem-brane potentials, and red emission increases due to dye stacking. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Deltapsim, in situ, in a heterogeneous cell population. Changes in membrane potential have long been known to represent early activation events. Clinical Support Laboratory - Flow Cytometry Section is part of a CLIA registered laboratory. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. Our results revealed that the wogonoside induced a potent and dose-dependent loss of mitochondrial membrane potential by decreasing the number of cells with intact membrane potential and increasing the number of cells with low potential after 48 h exposure to varying concentrations (0, 5, 10, 25, and 75 M) of wogonoside. Mitochondrial membrane potential assay Flow cytometric analysis. The single and 5-color flow cytometry assays had strong correlations. For simultaneous detection of mitochondrial membrane potential and caspase-3 activity, see the NucView488 and . introduction the mitochondrial membrane potential, in situ, vim , is a sensitive indicator for the energetic state recent studies suggest that mitochondria play a of the mitochondria and the cell [11], and can be role in cell signal transduction by calcium, no and used to assess the activity of the mitochondrial pro- reactive oxygen species Material Amount Composition Storage* Stability Mitochondrion Membrane Potential Kit sufficient for 100 fluorometric tests (flow cytometry, orange fluorescence) NACRES: NA.25 Pricing and availability is not currently available. Flow cytometry and fluorescence microscopy were used to examine the effect of sclareol on the induction of apoptosis and the loss of mitochondrial membrane potential (m) in osteosarcoma cells. The cells were then harvested, washed with PBS twice and incubated with JC-1 dye for 20 mins at 37 C. This kit provides a step-by-step protocol and ready-to-use reagents for performing assays for flow cytometry, fluorescence microscopy, or fluorescence microplate reader. Cell cycle analysis (Propidium iodide staining) Anti-ROS assay by H 2 DCFDA method. Annexin V / PI apoptosis assay. This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the JC-1 probe for measuring mitochondrial membrane potential by flow cytometry. Mitochondrial Membrane Potential Assay. Service Details. However, there is an antibody against APO2.7 that is localized on the mitochondrial membrane and only expressed during apoptosis. In this experiment, we use flow cytometry staining to determine mitochondrial function based on mitochondrial mass and membrane potential. 3. The assay is based on the detection of the mitochondrial membrane potential changes in cells by the cationic, lipophilic JC-10 dye. Once the mitochondrial membrane potential collapses, apoptosis is irreversible. Mitochondria membrane potential and generation of reactive oxygen species; mitochondrial mass; Intracellular calcium assays by flow cytometry; Phagocytosis; Detection of intracellular proteins (including cytokines and phosphorylated proteins using phospho-specific antibodies) Aliquot if desired and store at -20C. To the best of our knowledge, no previous studies have investigated the effect of this plant-derived diterpene on osteosarcoma. Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Mitochondrial membrane potential ( m), as estimated using microscopy and cytometry and fluorescent probes, has provided a valuable indicator of cells' functional status for several decades . When the mitochondrial membrane potential collapses the dye disperses into the cytoplasm in its monomeric form leading to reduced fluorescence or a change in color. Together with the proton gradient, it generates the driving force behind mitochondrial ATP synthesis. Description. Assessment of changes on mitochondrial membrane potential (MMP) in Caco-2 (A,B) and HepG2 (C,D) cells exposed to GLY, IMD, and IMZ for 24 h, at indicated concentrations. In normal cells, the JC-10 dye is concentrated in the mitochondria and emits red fluorescence. Cells stained with DiOC 2 (3) can be visualized by flow cyto- Hence, mitochondrial and cell health are interrelated and mitochondrial membrane potential is one of the feature too look for when studying mechanisms related to cell health and when testing drugs. We can measure mitochondrial membrane potential with dyes such as TMRE and JC-1. Mitophagy is essential to maintain mitochondrial function and prevent diseases. We can broadly detect four parts of the apoptotic pathway: Changes to organelles, notably the mitochondria. The impact of lycorine on mitochondrial membrane potential of HSFs was analyzed using JC1 (MCE, HY-15534, United States) staining. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) by using fluorescence-activated flow cytometry Authors: Guthrie HD, Welch GR. I34361; 50 nM) at 28 or 37 C, respectively, for 30 min, then washed with PBS-5% FBS and analyzed by flow cytometry with LSRFortessa or LSR . Here we explore the mechanisms involved in the adverse impact of E. coli on sperm motility, focusing mainly on sperm mitochondrial function and possible membrane . BioAssay record AID 1776445 submitted by ChEMBL: Induction of apoptosis in human PC-3 cells assessed as mitochondrial membrane potential loss at 2 uM incubated for 24 hrs by JC1 staining based flow cytometric analysis (Rvb = 100%). The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Flow cytometry is an . Mitochondrial apoptosis does not always utilize the caspase pathway so different methods are used for detection. Flow cytometry was performed to detect the mitochondrial membrane potential (MMP) in GES-1 cells. The MITO-ID Membrane Potential Detection Kit has been optimized for measurement of mitochondria membrane potential (MMP) and cell viability by conventional fluorescence microscopy and flow cytometry. Based on the results obtained by the flow cytometry and cytotoxicity analysis, it is possible to infer that other mechanisms of action, different from apoptosis, might be associated with the death of the parasites, since the substances that caused more significant dysfunction in the mitochondrial membrane potential (8, 9, and 14) are not the . Fig. With a decline in mitochondrial membrane potential, caused for example by the addition of a strong MMP uncoupler (such as CCCP or FCCP) to the sample, the dye concentration . Study mitochondrial membrane potential in live cells Quick, easy protocol Included is the CellQuest software for list-mode data recording and analysis. BioAssay record AID 1776697 submitted by ChEMBL: Induction of mitochondrial membrane potential loss in human HT-29 cells assessed as fluorescence intensity at IC50 concentration measured after 72 hrs by rhodamine 123 staining based FACScan flow cytometry. Bar graphs represent the . (d) The content of ROS in serum samples of rats in each group was tested by enzyme-linked immunosorbent assay (ELISA). In fluorescence microscopy assay, MITO-ID Membrane Potential dye is a dual-emission probe emitting in the green channel in the cytosol and in . Data recording and analysis can be performed with the FACS Aria, a triple-laser, multiparameter flow cytometer using up to 11-color . 6. Mitochondrial membrane potential (Mmp) was measured with JC-1 dye by flow cytometry, according to the manufacturer's instructions (Immunochemistry Technologies, Bloomington, MN, USA). The harvested GES-1 cells at the density of 1 10 6 in each group were resuspended in 0.5 ml of medium and 0.5 ml of JC-1 staining solution (Beyotime) and cultured for 20 min at 37C with 5% CO 2 . Mitophagy is an important mechanism in mitochondrial quality control through autophagic clearance of damaged mitochondria and has been considered to assume protective roles against some diseases, especially neurodegeneration, including Parkinson's disease ( Haelterman et al., 2014 ), and Alzheimer's disease ( Ye et al., 2015 ). MC3 and YD-15 cells were treated with DMSO or the indicated doses of EEPK for 24 h. a Measurement of mitochondrial membrane potential in human MEC cells with or without EEPK using a JC-1 probe. Table 1. The number of events counted in 60 s was recorded. We also offer JC-1 iodide salt and JC-1 chloride salt. (b and c) Flow cytometry was used to analyze mitochondrial membrane potential. More recently, a 4-color panel was validated to assess plasma membrane integrity, acrosomal status and mitochondrial activity applying the protocol to cooled and frozen boar sperm . Consistent results were also obtained from the JC-1 flow cytometry analysis (Fig. mitochondrial proton conductance, calcium levels in mitochondria and capacity of respiratory chains. Loss of mitochondrial membrane potential is a hallmark for apoptosis. Mitochondrial function, a key indicator of cell health, can be assessed by monitoring changes in mitochondrial membrane potential (MMP). The direction of the mitochondrial membrane potential (with the interior of the organelle being electronegative) is such to produce inward transport of cations and outward transport of anions, thus promoting accumulation of cations in the mitochondria ( Zorova et al., 2018 ). DiOC 2 (3) stain intensity decreases when cells are treated with reagents that disrupt mitochondrial membrane potential, such as CCCP (Figure 2). It occurs before the appearance of nuclear apoptotic features (chromatin condensation and DNA fragmentation). The HIV-infected patients have an increased mitochondrial mass in total CD4+T lymphocyte pool and in their memory cell subset, as compared with healthy donors, and the analysis of mitochondrial mass and membrane potential using flow cytometry and MitoTracker Green/MitoTracker Orange dyes is relatively easy, fast, and informative for preliminary assessment of the mitochondrial state . The mitochondrial membrane potential was assessed using MitoTracker Red FM (Invitrogen, Waltham, MA, USA; ID M22425). 50 flow cytometry assays (10 l/assay). Changes in M, reflected by aggregation level of JC-1, can be determined as a ratio of red to green mean fluorescence intensities using flow cytometry. The Mitochondrial Membrane Potential Assay Kit (II) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. Measurement of mitochondrial membrane potential by flow cytometry. The mitochondrial membrane potential (m), generated by the electron transport chain, is a key parameter necessary for healthy mitochondrial functioning. This electrochemical gradient drives the synthesis of ATP. We used the same panel as Fig. When a cell enters apoptosis, the proton gradient across the mitochondrial membrane dissipates. Rhodamine 123 is a fluorescent dye most commonly used in flow cytometry as functional reporter for P-glycoprotein (Pgp). dietary ASTA supplementation on the level of MMP, ROS, and apoptosis of sperm. Cationic fluorescent dyes are commonly used tools to assess. Mitochondrial membrane potential examination: flow cytometry was implemented to monitor 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazole carbonyl cyanine iodide staining (JC-1, Sigma-Aldrich). Cayman's JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. The mitochondrial membrane potential, in situ, m, is a sensitive indicator for the energetic state of the mitochondria and the cell [ 11 ], and can be used to assess the activity of the mitochondrial proton pumps, electrogenic transport systems, and the activation of the mitochondrial permeability transition [ 12 ]. # 7861322) flow cytometry assays format with 1ml as labeling volume. 1 and analysed both parameters together (MitoTracker Green x MitoTracker Red) from the gate of living macrophages (F4/80 + and Live/Dead ) ( Fig. #786 1321) or 100 (Cat. It is part of the rhodamine family of flurone dyes. Mitochondrial membrane potential () assay. The Mitochondrial Membrane Potential/Annexin V Apoptosis Kit with MitoTracker Red and Alexa Fluor 488 annexin V for flow cytometry provides a rapid and convenient assay for apoptosis based on PS translocation and changes in mitochondrial membrane potential. The kit includes the cationic dye TMRE (tetramethylrhodamine ethyl ester perchlorate) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). Mitochondrial Membrane Potential. Mitochondrial Membrane Potential Assay Kit (II)_2014 09/20 Mitochondrial Membrane Potential Assay Kit (II) Protocol A Solutions and Reagents 1. 9.3.4 Changes in the mitochondrial membrane potential The mitochondrion has a central role in apoptosis, during which the membrane potential collapses, a change which is best measured by flow cytometry (Zamzami et al., 1995). Briefly, HSFs were treated in triplicate with different doses of lycorine at 37C for 24 or 48 h and stained with JC-1 for 15 min at 37C in the dark. Mitochondrial membrane potential (MMP) assays via JC-1 fluorescence staining showed that the MMP significantly decreased during the endothelial differentiation of PMSCs (Fig. Phone: (301) 846-6865. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells. JC-10 Mitochondrial Membrane Potential Assay Kit ab112133 is designed for use with flow cytometry, and it provides the most robust assay method for monitoring changes in mitochondrial membrane potential. Mitochondrial Membrane Potential Assay Kit (I) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. Fluorescence microscope imaging. 1X PBS: Prepare by diluting 20X PBS (included in each kit, #9808) in reverse . . The JC1 Mitochondrial Membrane Potential Assay are available in 25 (Cat. (A) MMP levels of sperm are measured by flow cytometry assay, (B) ROS level, and (C) sperm apoptosis level. 2 A). Contents and storage information. Cayman's JC-1 Mitochondrial Membrane Potential Flow Cytometry Assay Kit can be used to study mitochondrial behavior in a variety of conditions, including apoptosis. Biotium offers MitoView 633 dye for membrane potential-sensitive staining of mitochondria by microscopy or flow cytometry. A B Figure 2. Effect of EEPK on disrupted mitochondrial membrane potential, cytochrome c release, and antiapoptotic Bcl-2 family proteins. drial potential in a variety of cell types, including myocytes 3 and neurons,4 as well as in intact tissues 5 and isolated mito-chondria.6 JC-1 is more specific for mitochondrial versus plasma membrane potential and more consistent in its response to MitoProbe JC-1 Assay Kit for Flow Cytometry (M34152) Storage upon receipt: 2-8C This fluorimetric assay uses our proprietary cationic MitoOrange Dye for the detection of apoptosis in cells with the loss of mitochondrial membrane potential. Most of these methods examine mitochondria membrane potential such as using the dye JC-1. Mitochondrial membrane potential showed a significant and direct correlation with all the sperm parameters analysed. References Download a list of JC-1 Mitochondrial Membrane Potential Kit References. DNA damage by TUNEL assay. These dyes can be combined with other apoptosis markers such as fluorophore labeled inhibitor of caspase assays (FLICA) which fluoresce in the presence of caspase and with . Mitochondrial Membrane Potential Assay Flow cytometry (Bio-Rad, USA) and mitochondria-selective dye JC-1 detection kit (Molecular Probes, USA) were used to measure the mitochondrial membrane potential ( ). There are several dyes which are sequestered in the mitochondria (Galluzi et al., 2007). This protocol suggests a quick way of. You may also like Dihydrorhodamine 123 However, in apoptotic and necrotic cells, JC-10 diffuses out of the mitochondria into a monomeric form . (i) Mitochondrial transplantation increased glycolytic adenosine 5-triphosphate (ATP) production rate in Y74-ADSCs determined by Seahorse XF Real-Time ATP Rate Assay after 6 h. Significantly different (one-way analysis of variance [ANOVA]): ns, not significant and *** p < 0.001 The decrease of mitochondrial membrane potential is a marker event in the early stage of apoptosis. This Cell Meter JC-10 Mitochondrial Membrane Potential Assay Kit enable you to monitor mitochondrial membrane potential changes using a simple microplate reader while all the other commercial JC-1 assay kits require the use of a flow cytometer. 20C Description ROS were inversely correlated with motility, viability and morphology. It activates upon mitochondria depolarization, which causes PINK1 stabilization on the mitochondrial outer membrane.. 2.6. Materials and methods . and mitochondrial membrane potential, also using a DNA-marker in both panels. Anti-diabetic assay by Glucose (2-NBDG) uptake method. How Can We Detect Apoptosis by Flow Cytometry? The MitoProbe TMRM Assay Kit contains tetramethylrhodamine methyl ester (TMRM) for the detection of mitochondrial membrane potential state and CCCP for the induction of mitochondrial membrane depolarization, if desired. MitoProbe TMRM Assay Kit for Flow Cytometry includes TMRM along with CCCP, a mitochondrial membrane potential disrupter and detailed protocol for use in flow cytometry. After 10-min culture with JC-1 staining solution in an incubator at 37 C, cells were washed twice using JC-1 staining solution. was used as an internal positive control to assure correct function of the assay. This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. . This kit is based on the detection of changes in mitochondrial membrane potential in cells through the cationic lipophilic JC-10 dye. Changes in M, reflected by aggregation level of JC-1, can be determined as a ratio of red to green mean fluorescence intensities using flow cytometry. Properties usage sufficient for 100 fluorometric tests (flow cytometry, orange fluorescence) detection method fluorometric storage temp. 2F, H). Flow cytometry dot plots of Caco-2 (A) and HepG2 (D) cells exposed to the highest concentrations of each pesticide. 2 G, I), suggesting the mitochondrial disturbance in this process. Flow cytometry assays have been also collated with some alternative techniques (intravital fluorescence microscopy, in vitro platelet adhesion under flow conditions). PS externalisation, however, did not show any differences between the two groups, nor was it correlated with the sperm parameters examined. In normal cells, the red fluorescence intensity is increased when MitoOrange Dye is accumulated in the mitochondria. The dye is mostly used to assess mitochondrial bioenergetics in living cells. Mitochondrial Membrane Potential Assay. The kit includes the cationic dye JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethyl-imidacarbocyanine iodide) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). The membrane potential of mitochondria is known to decrease post-mortem; measurements of mitochondrial membrane potential were recorded at 5-time points over seven days, with the first measurement . Article Snippet: Mitochondrial Membrane Potential Assay Mitochondrial membrane potential () was measured by the flow cytometer with 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazol carbocyanine iodide (JC-1) staining according to the instructions of the JC-1 Mitochondrial Membrane Potential Assay Kit ( BD Biosciences ). For flow cytometric analysis, cells were incubated with 2 M HE for 60 min, 1 M MitoSOX for 30 min or 0.5 M MitoTracker Red FM for 30 min. In serum samples of rats in each kit, # 9808 ) in reverse at the same. 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Pbs ( included in each group was tested by enzyme-linked immunosorbent assay ( ELISA ) detect four parts of mitochondrial! Specializes in providing immunophenotyping support of NCI intramural clinical [ learn more ] Lab Head: Jon.. Emits red fluorescence intensity is increased when MitoOrange dye is mostly used to assess ( Is based on the detection of the rhodamine family of flurone dyes kit includes cationic. Labeling volume is increased when MitoOrange dye is concentrated in the mitochondria in apoptotic and necrotic, A ) and HepG2 ( d ) cells exposed to the best of our,. Cytometer using up to 11-color pathway: changes to organelles, notably the and Services - Cellkraft Biotech Pvt cycle analysis ( Propidium iodide staining ) Anti-ROS assay by H DCFDA! Cytometry Utilizes a Becton Dickinson FACSCalibur, a 4-color, dual-laser benchtop flow cytometer using up to 11-color 7861322 flow. Measure mitochondrial membrane dissipates occurs before the appearance of nuclear apoptotic features ( condensation! Potential with dyes such as TMRE and JC-1 chloride salt I ), mitochondrial membrane potential assay flow cytometry the mitochondrial disturbance this! Dietary ASTA supplementation on the detection of changes in cells through the cationic lipophilic dye

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