Cmh 2 dcfda 5and6chloromethyl2,7dichlorodihydrofluorescein diacetate, acetyl ester is a derivative of dcfda, but with an additional thiol reactive chloromethyl group, which. Although application of nac, a well known antioxidant. Discover more with our introduction to flow cytometry. Oxiselect intracellular ros assay kit green fluorescence. Methods in molecular biology methods and protocols, vol 594. Resuspend stained cells in an appropriate volume of flow cytometry staining buffer. Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. Add fresh medium containing your drug of choice to the carboxyh 2 dcfda loaded cells and incubate as desired. Ros assay kit, uses 27dichlorofluorescin diacetate dcfhda, a cell. Flowjo basic tutorial download flowjo basic tutorial data download. Please see the productspecific flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended. 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. We stain primed bone marrowderived macrophages with these probes and induce ros production through fc. In this protocol, we applied h2dcfda to label the intracellular ros and detected the dcf intensity by flow cytometry.
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. Labeling of utricular sensory hair cells with the styryl. Functional assays byflow cytometry purdue university. Ros detection using dcfda jan102012 i am currently doing experiments about ros and cell signaling in two cancer cell lines. 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. Cells constantly generate reactive oxygen species ros during aerobic metabolism. Identification of ros using oxidized dcfda and flow cytometry article pdf available in methods in molecular biology clifton, n. 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. Cells flow single file through a flow cytometer and. Carter, and padmakumar narayanan cell function assays have been redefined over the past several years, largely because of technologies such as flow cytometry. Detection of intracellular reactive oxygen species cm. Introduction to flow cytometry flow cytometry is a popular laserbased technology.
The cell line i use is hct116, adherent human colon carcinoma cells. We have developed a simple, costeffective, and laborefficient twostep protocol for preparing adherent cells for highthroughput flow cytometry. 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. Presently, more than 40,000 journal articles referencing flow cytometry have been published. 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. Brdu staining kit for flow cytometry thermo fisher scientific. Your use of this pdf, the bioone complete website, and all posted and associated content indicates your. First, flow cytometry analyzes a population of cells on a cellbycell basis, a critical capability for todays. Identification of ros using oxidized dcfda and flow cytometry. This book provides instructions and guidelines for performing modern flow. 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 respiratory burst activity in. Production and detection of reactive oxygen species ros.
Principles and applications of flow cytometry table of. 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 and microscopy provide information on cell viability and the percent of null, low, and high ros producing cells. For flow cytometry follow the protocol for suspension cells up to point. Dcfda is typically excited with a 488 nm laser and emits in the fitc channel. The majority of flow cytometers are now digital systems. We utilize h2dcfda, a unique cellpermeable fluorogenic probe, compatible with. Flow cytometric detection of reactive oxygen species. The reduced nonfluorescent fluorescein h 2 dcfda can be oxidized and converted into fluorescent 2, 7dichlorofluorescein dcf by intracellular ros. 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. The goal of this laboratory demonstration and handson experiment is to obtain a clean. 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. Comprehensive and highly practical, flow cytometry protocols, second edition offers both basic and clinical scientists an uptodate collection of. So, fl1 is used in our protocol to collect green light corresponding to the oxidation of dichlorodihydrofluorescein dcf by ros.
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. Since the emission of the fluorescent dye is about 529nm, it can be measured in the fl1 green. Simultaneous analysis of reactive oxygen species and. 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. Determine fold change between control and treated samples. 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.
Based on a protocol developed by meike herget and zhaohua guo, edited by byron hartman and stefan heller for the bie course, woods hole. A vbottom, 96well dilution plate suspension cell protocol. 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. This process is performed at rates of thousands of cells per second. Reactive oxygen species ros detection kit promocell. Apr 20, 20 the reduced nonfluorescent fluorescein h 2 dcfda can be oxidized and converted into fluorescent 2, 7dichlorofluorescein dcf by intracellular ros. Does anyone have a flow cytometry protocol using dcfda. Performance characteristics adherent cells fluorescence microscopy nih3t3 and hela cells were cultivated under standard culture conditions and kept untreated or.
Twostep protocol for preparing adherent cells for high. Identification of ros using oxidized dcfda and flowcytometry article pdf available in methods in molecular biology clifton, n. Principles and applications of flow cytometry table of contents. The dcfda assay was carried out with flow cytometry according to the protocol described 27. 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. Flow cytometric analysis of isolated liver mitochondria to.
This indicator exhibits much better retention in live cells than h 2 dcfda. Labeling of utricular sensory hair cells with the styryl dye. Pdf cells constantly generate reactive oxygen species ros during. Exact concentrations must be determined on an individual basis by the end user. 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. Cmh 2 dcfda is a chloromethyl derivative of h 2 dcfda, useful as an indicator for reactive oxygen species ros in cells. Reactive oxygen species ros detection reagents see label. Flow cytometry protocol flow cell signaling technology. Flow cytometry protocol for direct staining of intracellular antigens and cytokines where the cells are permeabilised using leucoperm. Production and detection of reactive oxygen species ros in. Staining buffer and facs facs and if staining buffer with fbs mb boster technical blogs and news bosterbio flow cytometry protocol. Cells flow single file through a flow cytometer and multiple parameters can be collected and measured at a highspeed rate.
Detection protocols using dyes allow the experimenter to use various widely available methods such as microscopy, flow cytometry, plate reader etc. Additionally, this report provides a basic understanding of flow. Direct intracellular staining using leucoperm method biorad. Simultaneous analysis of reactive oxygen species and reduced. This information can be used to individually sort or separate subpopulations of cells. Cycling cells are incubated with 5bromo2deoxyuridine brdu, a synthetic analog of thymidine which incorporates into newly synthesized genomic dna during. The major difference between other techniques and those proposed using flow cytometry is the ability to. 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. The fluorescence generated is directly proportional to the amount of oxidized dcfda to dcf. Dilute tertbutyl hydroperoxide 55 mm to a concentration, in order to obtain a final. Flow cytometry flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering.
In this protocol, we applied h 2 dcfda to label the intracellular ros and detected the dcf intensity by 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 f. Analyze on flow cytometry using 485 nm laser for excitation and 535 nm for emission. This indicator exhibits much better retention in live cells than h. 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. The properties measured include a particles relative size, relative granularity or internal complexity, and relative.
This protocol assumes that the user is familiar with the principles and practices of flow cytometry and is able to run samples. These products are air sensitive and should be stored under dry argon or nitrogen. Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they. Flow cytometry is a widely used technique for identifying cell populations as well as measuring cell surface and intracellular molecules. 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. 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. 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. 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.
Ros detection cellbased assay kit dcfda cayman chemical. The exact concentration of dcfda required will depend on thecell line being used but a general starting range would be 1025. This is the job of the signal processing electronics. Ros detection using dcfda flow cytometry protocol online. Identification of ros using oxidized dcfda and flowcytometry. Pdf identification of ros using oxidized dcfda and flowcytometry.
Evaluation of reactive oxygen species ros generation in. Flow cytometer readouts transfer cells to tubes appropriate for your flow cytometer. 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 enhances the ability of the compound to bind to intracellular components, thereby prolonging the dyes cellular retention. Alternatively, a flow cytometer equipped with a blue laser 488 nm and a filter. Dcf is a highly fluorescent compound which can be detected by fluorescence spectroscopy with excitation emission at 485 nm 535 nm.
Exact concentrations must be determined on an individual. Flow cytometric analysis of isolated liver mitochondria has been described previously by several investigators. For microplate and flow cytometer read assays, determine ros. Nov 21, 2011 add fresh medium containing your drug of choice to the carboxyh 2 dcfda loaded cells and incubate as desired. Pdf identification of ros using oxidized dcfda and flow. 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. The assay uses the cell permeant reagent 2,7 dichlorofluorescin diacetate dcfda, a fluorogenic dye that measures. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry. Flow cytometry offers three important capabilities to researchers and clinicians. The following protocol demonstrates the use of a commercially available kit employing these 2 probes designed to measure ros by flow cytometry. Flow cytometric measurement of ros production in macrophages.