Flow cytometry protocol pdf flow cytometry combines cell biology with the study of light waves and employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. Detect mitochondrial depolarization with the mitopt tmrm assay. Use this 10 m working solution to stain cells using any of the three protocols described in this kit insert. Thus, our protocol can also be used to compare the mitochondrial membrane potential of. Flow cytometry is a very useful tool to simultaneously analyze several. We have recently developed a number of protocols that permit the flow cytometric analysis of mycoplasmas for the purposes of detection, enumeration and viability determination. Livedead stains are useful probes to include when analyzing cell surface protein expression by flow cytometry, because they allow intracellular fluorescence signal from dead cells with permeable plasma membranes to be excluded from analysis. Current protocols in cytometry wiley online library.
Loss of mitochondrial membrane potential is indicative of apoptosis. Design and optimization protocol for highdimensional. Principle of flow cytometry the basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted and sorted. Mitoprobe tmrm kit for flow cytometry 3 methods prepare reagent mitoprobe tmrm reagent is provided as a x stock solution at a concentration of 2. Carbonyl cyanide 4trifluoromethoxy phenylhydrazone fccp. Flow cytometric analysis of tmre staining in jurkat cells.
Wash the cells by adding 1 ml or more if many samples of 1x pbs to the flow tube. Mitoprobe tmrm kit for flow cytometry thermo fisher scientific. Cell preparation for flow cytometry thermo fisher scientific. Topics shaprio, practical, flow, cytometry, download, practical flow cytometry, free, online collection. Here we describe a modified protocol for accurate m measurement by tmrmbased flow cytometry which corrects for xenobiotic transporter. The tmrm dye has a delocalized positive charge dispersed throughout its molecular structure. Bd mitoscreen flow cytometry mitochondrial membrane. Flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death. Ac jc1 fluorescence is seen in both the fl2 and fl1 channels r1 in the control untreated cell. Tmre emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of tmre fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low m. As a cationic and lipophilic fluorescent dye, tmrm can be used to detect.
Dye preparation bring bd pharmingen mitostatus tmre dye powder and fresh cell culturegrade dimethyl sulfoxide dmso. Spin down cell suspension at rpm for 5 minutes and decant supernatant. This process is performed at rates of thousands of cells per second. Flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Detection of mitochondrial mass, damage, and reactive oxygen species by flow cytometry article pdf available in cold spring harbor protocols 20159. Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. This retains the target protein in the original cellular location. When accumulated in a negatively charge polarized mitochondria, tmrm fluoresces orange. Liveordye fixable viability staining kits are designed for discrimination between live and dead cells during flow cytometry. Staining of intracellular antigens for flow cytometry protocols depends on various fixation and permeabilization methods to allow access of antibodies to internal cellular proteins. Thus, peripheral blood cells or cells that grow in suspension are well suited for analysis by flow cytometry. Flow cytometric analysis of the cadmiumexposed green alga.
Dead cells can compromise the integrity of the data by nonspecifically binding antibodies. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. Presently, more than 40,000 journal articles referencing flow cytometry have been published. In this ros protocols article, we described the flow cytometric protocol involving the use of various concentrations of mitosox 1, 2. These are presented here in the hope that they will be of use to the wider flow cytometry community. Flow cytometric analysis of the cadmiumexposed green alga chlamydomonas reinhardtii chlorophyceae. 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, m, in situ, in a heterogeneous cell population. Viability staining is an essential component of any flow cytometry experiment. Flow cytometry facs protocols psr the bd facscalibur platform allows users to perform both cell analysis and cell sorting in a single benchtop system. Flow cytometry intracellular staining protocol fix cells before intracellular staining to fixing and permeabilization ensure stability of soluble antigens or antigens with a short halflife see the special recommendations below for exceptions. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Assessing mitochondrial redox status by flow cytometric. This protocol can be used to determine the purity of the mitochondrial preparation, to detect calci.
Tmremitochondrial membrane potential assay kit ab1852 is used for quantifying changes in mitochondrial membrane potential in live cells by flow cytometry, microplate spectrophotometry and fluorescent microscopy. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. A successful staining procedure in all cases is dependent on optimization of experimental conditions through titering of. The system supports a wide variety of research and clinical applications and is complemented by a broad suite of intuitive software solutions to. Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. We also compared the mitosoxbased flow cytometry with lucigeninderived chemiluminometry for. Tmrm staining is used to monitor mitochondrial function. Protocols for the flow cytometric analysis of mycoplasmas. The technology can provide rapid, quantitative, multiparameter analyses on single living or dead cells based on the measurement of visible and. Quantitative assay by flow cytometry of the mitochondrial.
Add an appropriate volume of staining buffer generally 50 ul per 1 x 10e6 cells. Bd pharmingen mitostatus tmre tetramethylrhodamine ethyl ester is a fluorescent dye. Therefore the recommended staining period for tmrm is 1 h rather than 30 min as mentioned in one tmrm manufacturers protocol. Protocols using mitopt tmre or tmrm at concentrations 50 nm should generally include a single wash step to minimize background fluorescence issues. The mitopt m 500 test kit contains enough tmre or tmrm reagent to evaluate 500 flow cytometry or fluorescence microscopy cell samples at a 200 nm dye concentration. Pdf measurement of mitochondrial mass by flow cytometry. Can be used for the live cell dynamic study of mitochondrial membrane potential. Tmre mitochondrial membrane potential assay gbiosciences. Tmrm signal can be detected with fluorescence microscopy, flow cytometry, cell sorting, high throughput screening, and high content analysis. Principles of the flow cytometer flow cytometry basics guide 3 1 principles of the flow cytometer fluidics system one of the fundamentals of flow cytometry is the ability to measure the properties of individual particles. M for detecting mitochondrial ros in control and mitochondrial dnadeficient md melanoma b16f10 cells. Note the optimal cell density should be determined specifically for each.
Assessing mitochondrial redox status by flow cytometric methods. Reconstitute tmre dye powder in dmso at a stock concentration of 0. Tmretmrm mitochondrial membrane potential assessment. Tetramethylrhodamine tmrm thermo fisher scientific. An overview of the stepbystep protocol that illustrates the design of a highdimensional spectral flow cytometry panel. Tmrm assay kit mitochondrial membrane potential ab228569 is designed to quantify changes in mitochondrial membrane potential in living cells using flow cytometry, fluorescence plate reader, or fluorescence microscopy.
Icts mitopt tmrm assay utilizes the potentiometric fluorescent dye tmrm to detect mitochondrial membrane permeability and membrane depolarization. This information can be used to individually sort or separate subpopulations of cells. Facs flow cytometry is a technique used to measure the physical and chemical properties of cells orthe physical and chemical properties of cells or. Fluorescence spectra of mitoprobe tmrm assay kit for flow cytometry. Isolation of pbmc from whole blood introduction singlecell suspensions are required for all flow cytometry assays. Improving the accuracy of flow cytometric assessment of. Other applications flow cytometry is indicated in the evaluation of serous effusions and csf, including aqueous or vitreous humor of patients with a history of hematolymphoid neoplasia. When tmrm dye levels have not yet reached perfect equilibrium, unexpected changes in staining intensity can be detected during data acquisition in the flow cytometry process. In a healthy cell with active mitochondria, tmrm is readily sequestered, thereby emitting a redorange fluorescent signal. One of the most exciting applications of miniaturized chip.
Flow cytometry tested during development bioimaging tested during development recommended assay procedure. Cell preparation for flow cytometry research use only. Tmre is suitable for staining mitochondria in live cells only. Flow cytometric analysis of isolated liver mitochondria to. Our data suggest that nao may be more suitable than mtg for assessment of mitochondrial mass by flow cytometry during oxidative stress. Life cell quantification of mitochondrial membrane potential at the. Tmrm signal can be detected with fluorescence microscopy, flow cytometry, cell. Quick, easy protocol determining the state of mitochondrial health can be achieved by detecting changes in the mitochondrial membrane potential. Flow cytometry is a rapid and useful method of obtaining a diagnosis. Here, we aimed to develop an automated protocol allowing sensitive quantifi cation of dw in living cells. D graphic representation of cell death expressed as a % of annexin v fitc and dapipositive dead cells, and corrected for. Cuttingedge and comprehensive, flow cytometry protocols, fourth edition is a valuable resource for researchers and scientists who are interested in continuing andor expanding their knowledge of flow cytometry. Principles and applications of flow cytometry table of.
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