Flow cytometer readouts transfer cells to tubes appropriate for your flow cytometer. Refinement of the dichlorofluorescein assay for flow cytometric. These reagents can be detected using the appropriate benchtop instrument such as the attune acoustic focusing cytometer, tali imagebased cytometer, and floid cell imaging station see table 2, page 2. Dilute tertbutyl hydroperoxide 55 mm to a concentration, in order to obtain a final.
Does anyone have a flow cytometry protocol using dcfda. Proceed to step 8 for purified or biotinylated primary antibodies. The exact concentration of dcfda required will depend on thecell line being used but a general starting range would be 1025. The properties measured include a particles relative size, relative granularity or internal complexity, and relative.
Assess ros by immediately analyzing your cells by flow cytometry using the fl1 channel green fluorescence, or by fluorescence plate reader, or by fluorescence microscopy. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry. Brdu staining kit for flow cytometry thermo fisher scientific. Principles and applications of flow cytometry table of contents. Identification of ros using oxidized dcfda and flow cytometry article pdf available in methods in molecular biology clifton, n.
Dcf is a fluorescent compound which can be detected by fluorimeter, flow cytometer or fluorescence microscope with a maximum excitation and emission spectra of 485 nm and 535 nm. Exact concentrations must be determined on an individual. Flowjo basic tutorial download flowjo basic tutorial data download. The major difference between other techniques and those proposed using flow cytometry is the ability to. This book provides instructions and guidelines for performing modern flow. Flow cytometry dcfda cellular reactive oxygen species detection assay kit ab1851 ab1851 dcfda labeled and unlabeled jurkat cells were treated with 50 m tertbutyl hydrogen peroxide tbhp, then analyzed by flow cytometry. A representative whole lung flow cytometry plots to depict the gating strategy for il12byet40 homozygous mice il12p40ireseyfp reporter at 3 or 5 dpi with h5n1vnpr8. Flow cytometry and microscopy provide information on cell viability and the percent of null, low, and high ros producing cells. We have developed a simple, costeffective, and laborefficient twostep protocol for preparing adherent cells for highthroughput flow cytometry.
Cycling cells are incubated with 5bromo2deoxyuridine brdu, a synthetic analog of thymidine which incorporates into newly synthesized genomic dna during. Flow cytometric detection of reactive oxygen species bioprotocol. In this protocol, we applied h2dcfda to label the intracellular ros and detected the dcf intensity by flow cytometry. Pdf cells constantly generate reactive oxygen species ros during. The majority of flow cytometers are now digital systems. This process is performed at rates of thousands of cells per second. If a vbottom 96well plate or centrifuge microplate adapter is unavailable, volumes found in this protocol are applicable to 1. Production and detection of reactive oxygen species ros in. Labeling of utricular sensory hair cells with the styryl. After diffusion in to the cell, dcfda h2dcfda dcfhda is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ros into 2, 7 dichlorofluorescein dcf. Flow cytometric analysis of respiratory burst activity in. These products are air sensitive and should be stored under dry argon or nitrogen.
Although application of nac, a well known antioxidant significantly counteract the effect of my chemical, i always suffer one problem that the chemical shows no obvious influence on ros level when using dcfda or dhe. The following protocol demonstrates the use of a commercially available kit employing these 2 probes designed to measure ros by flow cytometry. Methods in molecular biology methods and protocols, vol 594. Cellular ros levels can be measured in live cells by a technique that converts 2,7 dichlorofluorescin diacetate dcfda which is oxidized to a fluorescence dye 2, 7 dichlorofluorescein dcf. The fluorescence generated is directly proportional to the amount of oxidized dcfda to dcf. Presently, more than 40,000 journal articles referencing flow cytometry have been published. Add fresh medium containing your drug of choice to the carboxyh 2 dcfda loaded cells and incubate as desired. Flow cytometric analysis of isolated liver mitochondria has been described previously by several investigators. Flow cytometry is a widely used technique for identifying cell populations as well as measuring cell surface and intracellular molecules. In this protocol, we applied h 2 dcfda to label the intracellular ros and detected the dcf intensity by flow cytometry. Introduction to flow cytometry flow cytometry is a popular laserbased technology. Resuspend stained cells in an appropriate volume of flow cytometry staining buffer.
Reactive oxygen species ros detection reagents see label. Evaluation of reactive oxygen species ros generation in. Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. Direct intracellular staining using leucoperm method biorad. Dcfda is typically excited with a 488 nm laser and emits in the fitc channel. The dcfda assay was carried out with flow cytometry according to the protocol described 27. Membrane potential was measured using rhodamine123 rh123 717, 3,3 dihexyloxacarbocyanine iodide dioc 6 3 18,19, or 5,5,6,6 tetrachloro1,1,3,3 tetra. Simultaneous analysis of reactive oxygen species and. The ebioscience brdu staining kit for flow cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells by flow cytometric analysis. Flow cytometry protocol flow cell signaling technology.
Cmh 2 dcfda is a chloromethyl derivative of h 2 dcfda, useful as an indicator for reactive oxygen species ros in cells. Ros induces the modification of carboxyh 2 dcfda that fluoresces green as detected by flow cytometry, the fluorescent peak in h 2 o 2 treated cells shift compared to the peaks in controls h 2 o 2 treated cells stained with oxidation insensitive dye and nontreated cells stained with carboxyh 2 dcfda. Cells constantly generate reactive oxygen species ros during aerobic metabolism. Grow cells stain cells with dcfda at 37c measure fluorescence exem 485535 nm in a microplate reader or flow cytometer, or image with a fluorescence microscope. Your use of this pdf, the bioone complete website, and all posted and associated content indicates your. Ros detection using dcfda jan102012 i am currently doing experiments about ros and cell signaling in two cancer cell lines.
Based on a protocol developed by meike herget and zhaohua guo, edited by byron hartman and stefan heller for the bie course, woods hole, august 2011. The assay uses the cell permeant reagent 2,7 dichlorofluorescin diacetate dcfda, a fluorogenic dye that measures. Carter, and padmakumar narayanan cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry. Pdf identification of ros using oxidized dcfda and flowcytometry. Thanks a lot, good luck with your flow cytometry sure, although id recommend to check whether you can adapt it to the cells you use. Exact concentrations must be determined on an individual basis by the end user. First, flow cytometry analyzes a population of cells on a cellbycell basis, a critical capability for todays. This information can be used to individually sort or separate subpopulations of cells. Ros detection using dcfda flow cytometry protocol online. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data.
Cmh 2 dcfda 5and6chloromethyl2,7dichlorodihydrofluorescein diacetate, acetyl ester is a derivative of dcfda, but with an additional thiol reactive chloromethyl group, which. There are also techniques for monitoring gene transfer and expression using fluorescent protein technology, highthroughput screening for discovery of novel protein interactions, phenotypic and functional characterization of tcell subsets and precursors, and microbial flow cytometry, to highlight but some of the many useful procedures. 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 f. Fcm fixation buffer 10x is intended for preservaton of human and rodent cells cultured or from a blood sample prior to permeabilization with fcm permeabilization buffer and subsequent. Ros induces the modification of carboxyh 2 dcfda that fluoresces green as detected by flow cytometry, the fluorescent peak in h 2 o 2 treated cells shift compared to the peaks in controls. Oxiselect intracellular ros assay kit green fluorescence. Analyze on flow cytometry using 485 nm laser for excitation and 535 nm for emission. After diffusion in to the cell, dcfda h2dcfda dcfhda is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ros into 2, 7 dichlorofluorescein. Since the emission of the fluorescent dye is about 529nm, it can be measured in the fl1 green. This protocol assumes that the user is familiar with the principles and practices of flow cytometry and is able to run samples. Cells flow single file through a flow cytometer and multiple parameters can be collected and measured at a highspeed rate. The reduced nonfluorescent fluorescein h 2 dcfda can be oxidized and converted into fluorescent 2, 7dichlorofluorescein dcf by intracellular ros.
The cell line i use is hct116, adherent human colon carcinoma cells. For microplate and flow cytometer read assays, determine ros. Flow cytometry protocol for direct staining of intracellular antigens and cytokines where the cells are permeabilised using leucoperm. Please see the productspecific flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended. Apr 20, 20 the reduced nonfluorescent fluorescein h 2 dcfda can be oxidized and converted into fluorescent 2, 7dichlorofluorescein dcf by intracellular ros. We stain primed bone marrowderived macrophages with these probes and induce ros production through fc. Identification of ros using oxidized dcfda and flowcytometry. Functional assays byflow cytometry purdue university. Although application of nac, a well known antioxidant. Cmh 2 dcfda passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiolreactive chloromethyl group reacts with intracellular glutathione and other.
Principles and applications of flow cytometry table of. Flow cytometric measurement of ros production in macrophages. Additionally, this report provides a basic understanding of flow. Detection of intracellular reactive oxygen species cm. We utilize h2dcfda, a unique cellpermeable fluorogenic probe, compatible with. For flow cytometry follow the protocol for suspension cells up to point.
Performance characteristics adherent cells fluorescence microscopy nih3t3 and hela cells were cultivated under standard culture conditions and kept untreated or. Based on a protocol developed by meike herget and zhaohua guo, edited by byron hartman and stefan heller for the bie course, woods hole. Staining buffer and facs facs and if staining buffer with fbs mb boster technical blogs and news bosterbio flow cytometry protocol. So, fl1 is used in our protocol to collect green light corresponding to the oxidation of dichlorodihydrofluorescein dcf by ros. Twostep protocol for preparing adherent cells for high. Add 2 ml of flow cytometry staining buffer and centrifuge at 4006x g for 45 minutes at room temperature. This is the job of the signal processing electronics. Fcm fixation buffer 10x is intended for preservaton of human and rodent cells cultured or from a blood sample prior to permeabilization with fcm permeabilization buffer and subsequent intracellular staining with monoclonal or polyclonal fluorochromeconjugated antibodies. Cells flow single file through a flow cytometer and. Identification of ros using oxidized dcfda and flow cytometry. Flow cytometry flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. Flow cytometric detection of reactive oxygen species. Ros detection cellbased assay kit dcfda cayman chemical.
Cell biolabs oxiselect intracellular ros assay kit green fluorescence is a cellbased assay for measuring hydroxyl, peroxyl, or other reactive oxygen species activity within a cell. Flow cytometric analysis of isolated liver mitochondria to. Dcf is a highly fluorescent compound which can be detected by fluorescence spectroscopy with excitation emission at 485 nm 535 nm. Labeling of utricular sensory hair cells with the styryl dye. Cellular ros levels can be measured in live cells by a technique that converts 2,7 dichlorofluorescin diacetate dcfda which is oxidized to a fluorescence dye 2, 7. Flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. This indicator exhibits much better retention in live cells than h 2 dcfda. The staining workflow is simple figure 2, page 2, and the reagent can be applied to cells. Cmh 2 dcfda 5and6chloromethyl2,7dichlorodihydrofluorescein diacetate, acetyl ester is a derivative of dcfda, but with an additional thiol reactive chloromethyl group, which enhances the ability of the compound to bind to intracellular components, thereby prolonging the dyes cellular retention. A vbottom, 96well dilution plate suspension cell protocol. Pdf identification of ros using oxidized dcfda and flow. Nov 21, 2011 add fresh medium containing your drug of choice to the carboxyh 2 dcfda loaded cells and incubate as desired. Detection protocols using dyes allow the experimenter to use various widely available methods such as microscopy, flow cytometry, plate reader etc.
Reactive oxygen species ros detection kit promocell. Discover more with our introduction to flow cytometry. Alternatively, a flow cytometer equipped with a blue laser 488 nm and a filter. This indicator exhibits much better retention in live cells than h. Determine fold change between control and treated samples.
Flow cytometry offers three important capabilities to researchers and clinicians. Identification of ros using oxidized dcfda and flowcytometry article pdf available in methods in molecular biology clifton, n. The goal of this laboratory demonstration and handson experiment is to obtain a clean. Comprehensive and highly practical, flow cytometry protocols, second edition offers both basic and clinical scientists an uptodate collection of.
Combine the recommended quantity of each primary antibody in an appropriate volume of flow cytometry staining buffer so that the final staining volume is 100 l i. Ros assay kit, uses 27dichlorofluorescin diacetate dcfhda, a cell. Tissue culture vessels and appropriate culturing media. Simultaneous analysis of reactive oxygen species and reduced. Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they. Production and detection of reactive oxygen species ros.
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