Flow cytometry was employed to quantify mitosox red intensities specific for mto 2 oscillatory shear stress induced jnk activation fig. Storage and handling upon receipt, the mitosox red reagent should be stored upright, desiccated, and protected from light at. Therefore, quantification of mitosox fluorescence without verifying mitochondrial localization e. Cells were then treated for 4 hours with a titration series of. Flow cytometry facs protocols psr the bd facscalibur platform allows users to perform both cell analysis and cell sorting in a single benchtop system. Mitosoxbased flow cytometry for detecting mitochondrial ros.
A closer look at mitochondrial dysfunction with mitosox red. The xaxis denotes the mitosox red fluorescence detected in the 564606 nm spectral region and the yaxis denotes the number. Mitosox is designed to measure mitochondrial superoxide and other reactive oxygen species. Mitosox red reagent is a novel fluorogenic dye specifically targeted to mitochondria in live cells. While the optimal staining concentration and incubation times can vary by application, typical results are obtained by. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Whole blood samples may be used undiluted unless the cell count is high, e. Jc1 mitochondrial membrane potential assay kit ab1850. Preparation of cells for flow cytometry for further information. Readily oxidized by superoxide but not by other ros or rnsgenerating systems. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer. Jurkat cells tcell leukemia, human were treated with a pbs or b 200. Introduction to flow cytometry flow cytometry is a popular laserbased technology.
Assessing mitochondrial redox status by flow cytometric methods. The ros generation was assessed by confocal microscopy or flow cytometry after staining with mitosox red and. Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. Mitosox red mitochondrial superoxide indicator mw 759 is supplied in 10 vials, each containing 50. The production of superoxide by mitochondria can be measured using mitosox red.
Invitrogen mitosox red mitochondrial superoxide indicator. Immunophenotyping is the analysis of heterogeneous populations of cells using labeled. Application of imaging flow cytometry for the characterization of intracellular attributes in chinese hamster ovary cell lines at the singlecell level. The following protocol demonstrates the use of a commercially available kit employing these 2 probes designed to measure ros by flow cytometry. The cellpermeable cellrox deep red reagent is essentially nonfluorescent while in a. Using a flow cytometer, mitosox red was excited at 488 nm and. Assay of mitochondrial levels of reactive oxygen species. It is recommended that experimental conditions, such as antibody concentration, incubation time, and temperature, be optimized for each flow cytometry experiment. This book provides instructions and guidelines for performing modern flow cytometry. The images are background is subtracted, and cellfree areas, debris and dead cells are masked. Mitosox red is a dye used to measure superoxide production in the mitochondrial.
Mitosox red reagent is novel fluorogenic dye specifically targeted to mitochondria in live cells. This protocol was developed using live bovine pulmonary epithelial. Please see the end of this protocol for recipes indicated by. Highthroughput analysis of cho cells with flow cytometry, abstract. Labels, dyes, and stains can be used for multiparametric analysis understand more properties about a cell. Flow cytometry uses the light properties scattered from cells or particles for identification or quantitative measurement of physical properties.
In this ros protocols article, we described the flow cytometric protocol. Proliferation stimulation promotes b cell activation and species in. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c. We also compared the mitosox based flow cytometry with lucigeninderived chemiluminometry for. In the mitochondria, the probe is oxidized by superoxide, resulting in the emission of red fluorescence. Does anyone have good flow cytometry protocol for mitosox to. Once in the mitochondria, mitosox red reagent is oxidized by superoxide and exibits red. Detection of mitochondrial mass, damage, and reactive oxygen species by flow cytometry article pdf available in cold spring harbor protocols 20159.
Mitosox red mitochondrial superoxide indicator, for live. Robust assays can detect and monitor multiple analytes to produce a tremendous amount of data while conserving sample. The flow cytometry protocols below provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer. Flow cytometry analysis of intracellular mitosox red f. Assessing mitochondrial redox status by flow cytometric. The cellrox deep red flow cytometry assay kit is specifically formulated for flow cytometry and provides the key reagents necessary for the detection of ros in live cells. Robust assays can detect and monitor multiple analytes to produce a. Oxidative damage applications a common application of mitosox red indicator is the.
Facs is an abbreviation for fluorescenceactivated cell sorting, which is a flow cytometry technique that further adds a degree of functionality. This is one of the simplest and most common staining methods. Like the mitotracker probes, mitosox red is taken up passively by cells. Does anyone have good flow cytometry protocol for mitosox. Cellrox deep red flow cytometry assay kit 3 figure 3 cellrox deep red reagent can be used in conjunction with the sytox blue dead cell stain to differentiate live stressed cells from dead cells. Reactive oxygen species produced up or downstream of calcium. We stain primed bone marrowderived macrophages with these probes and induce ros production through fc.
Mitosox red mitochondrial superoxide indicator for live. This protocol shows for the first time that it is possible to measure mitochondrial ros content in different lymphocyte populations, and can be applied to. Incubate for 5 min at room temperature fixed cells or on ice live cells. Figure 4 from mitosoxbased flow cytometry for detecting. Immunodetection of outer membrane proteins by flow. Fab92, and mitosox red fluorescence was recorded at 561 nm at 5min intervals over 30.
Availability of different conjugates and putting the. Flow cytometry is a popular cell biology technique that utilizes laserbased technology to count, sort, and profile cells in a heterogeneous fluid mixture. M tbhp for 30 minutes before labeling with the cellrox deep red flow cytometry. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in flow cytometry. Mitosox red mitochondrial superoxide indicator for livecell. The system supports a wide variety of research and clinical applications and is complemented by a broad suite of intuitive software solutions to. Flow cytometry analysis of intracellular mitosox red f openi. Detection of mitochondrial mass, damage, and reactive. We stain primed bone marrowderived macrophages with. Mitosox red mitochondrial superoxide indicator thermo fisher. The selective detection of mitochondrial superoxide by. The selective detection of mitochondrial superoxide by live. M for detecting mitochondrial ros in control and mitochondrial.
Flow cytometry fcm is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles in this process, a sample containing cells or particles is. Once in the mitochondria, mitosox red reagent is oxidized by superoxide and exibits red fluorescence absorptionemission maxima. Multicolor flow cytometry panels multicolor flow cytometry is a powerful tool enabled by development of advanced flow cytometers. Abs, flow cytometry analysis, and b cell purification. Quantification of mitochondrial reactive oxygen species in. A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine he or its mitochondrially targeted. M for detecting mitochondrial ros in control and mitochondrial dnadeficient md melanoma b16f10 cells. Oxidation of mitosox red reagent by superoxide produces red fluorescence.
Detection of mitochondrial mass, damage, and reactive oxygen. This is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens. Wash cells twice with pbs or hbss, spinning the tube at 300 x g for 5 minutes. Methods to monitor ros production by fluorescence microscopy. Flow cytometry protocol for staining membraneassociated. Reactive oxygen species produced up or downstream of. Detection of mitosox red indicator by flow cytometry typically uses the fl2 emission channel 58542 nm. Pdf detection of mitochondrial mass, damage, and reactive. In this ros protocols article, we described the flow cytometric protocol involving the use of various concentrations of mitosox 1, 2. In this protocol, method and experimental design will be provided to demonstrate that 1. To make a 1 mm stock solution, reconstitute the solid in 94. Mitotracker red cmxros, mitotracker red fm, and mitotracker deep red fm are all well suited for multicolor labeling experiments because their red fluorescence is well resolved from the green fluorescence of other probes. Technical bulletin,application note flow cytometry created date.
We also compared the mitosoxbased flow cytometry with lucigeninderived chemiluminometry for. Mitochondria are isolated from rodent spinal cords and subjected to a. This protocol assumes that the user is familiar with the principles and practices of flow cytometry and is able to run samples according to the operators. Discover more with our introduction to flow cytometry. The xaxis denotes the mitosox red fluorescence detected in the 564606 nm. Facs is an abbreviation for fluorescenceactivated cell.
Each vial of mitotracker green fm contains 50 g lyophilized solid. To make a 1 mm stock solution, reconstitute the solid in 74. Flow cytometry protocols flow cytometry guide biorad. M for detecting mitochondrial ros in control and mitochondrial dnadeficient md melanoma b16. This book provides instructions and guidelines for performing modern flow cytometry that should render experiments that are more successful. Interleukin6 counteracts therapyinduced cellular oxidative stress in. Flow cytometric measurement of ros production in macrophages. Mitosoxbased assays are widely used to detect mitochondrial reactive oxygen species ros, especially superoxide. Protocol detection of mitochondrial mass, damage, and reactive oxygen species by flow cytometry daniel puleston cold spring harb protoc. Low micromolar concentrations of the superoxide probe mitosox. Low micromolar concentrations of the superoxide probe. M dcfhda fl1 or mitosox red for 15 min at 37 c in a final volume of 450. Here, we have applied this protocol based upon multi.
Using mitosox red, it is also possible to analyze the production of the mitochondrial superoxide anion. Mitosox red reagent is livecell permeant and rapidly and selectively targeted to the mitochondria. While the optimal staining concentration and incubation times can vary by application, typical results are obtained by diluting the stock solution directly into growth. Flow cytometry is the main technology that generates multiple information at the. The fluorescence resulting from cellrox oxidative stress reagents can be measured using traditional fluorescence microscopy, highcontent imaging and analysis, microplate fluorometry, or flow cytometry. Storage and handling upon receipt, the mitosox red reagent should be stored upright. Oxidative damage applications a common application of mitosox red indicator is the correlation of superoxide generation with the accumulation of oxidative damage products such as lipid peroxides and modified dna bases such as 8oxodg. Mitosox based assays are widely used to detect mitochondrial reactive oxygen species ros, especially superoxide. Aug 30, 2016 here, we have applied this protocol based upon multi. Flow cytometry protocol flow cell signaling technology. Mitosoxbased flow cytometry for detecting mitochondrial. A general protocol is described to improve the specificity for imaging superoxide formation in live cells via fluorescence microscopy with either hydroethidine he or its mitochondrially.
Does anyone have good flow cytometry protocol for mitosox to check ros production in c. Flow cytometry clearly represents the most rapid and powerful tool for investigating ros at the singlecell level, allowing the generation of multiple information on single cells in a highthroughput manner, with. A closer look at mitochondrial dysfunction with mitosox. Quantification of mitochondrial reactive oxygen species in living. Flow cytometry is used for research applications such as immunophenotyping, dna studies, cell cycle analysis, and fluorescenceactivated cell sorting facs. Flow cytometry analysis of intracellular mitosox red fluorescence from live hela cells incubated at 37c for 60 h in. In this ros protocols article, we described the flow cytometric protocol involving the. Flow cytometry clearly represents the most rapid and powerful tool for investigating ros at the singlecell level, allowing the generation of multiple information on single cells in a highthroughput manner, with high sensitivity and reproducibility 11,12. In modern flow cytometry, the way you start an experiment remains the same. Additionally, this report provides a basic understanding of flow cytometry. This protocol is designed for staining of cell surface proteins. Flow cytometry is now a widely used method for analyzing the. The production of intracellular ros was measured using the oxidationsensitive probe dcfhda and the mitochondriatargeting probe mitosox red by flow cytometry. These capabilities have made flow cytometry a powerful tool with multiple applications for researchers and clinicians alike.
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