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Molecular Probes™ CellROX™ Green Flow Cytometry Assay Kit

Catalog No. C10492
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C10492 100 assays
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Catalog No. C10492 Supplier Molecular Probes™ Supplier No. C10492
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Description

CellROX™ Green Flow Cytometry Assay Kit

The CellROX Green Flow Cytometry Assay Kit enables the flow cytometric detection of reactive oxygen species (ROS) in live cells. The kit includes the novel, fluorogenic CellROX Green Reagent, as well as SYTOX Red Dead Cell Stain, N-acetyl cysteine (an antioxidant, for negative control), and tert-butyl hydroperoxide solution (TBHP, an inducer of ROS).

The CellROX Green Flow Cytometry Assay Kit features:

• Fluorogenic probe formulated for flow cytometry that is oxidized in the presence of ROS
• Multicolor compatibility—minimal overlap with fluorophores excited by other laser lines, allowing easy multiplexing with other reagents
• Simple protocol—cells can be stained in complete media or other appropriate buffer, no need for serum free media

The CellROX Green Detection Reagent is cell-permeable and non-fluorescent or very weakly fluorescent while in the reduced state. Upon oxidation, the reagent binds DNA and exhibits a strong fluorogenic signal that has absorption/emission maxima of 508/525 nm and remains localized to the nucleus and mitochondria. When used together with the included SYTOX Red Dead Cell Stain, oxidatively stressed and nonstressed cells are reliably distinguished from dead cells by flow cytometry.

Specifications

Format Tube
Quantity 100 assays
Excitation/Emission CellROX green: 508/525, SYTOX red: 640/658

Frequently Asked Questions (FAQs)

What are the fluorescence excitation/emission maxima for the CellROX Green Reagent provided in the CellROX Green Flow Cytometry Assay Kit?

The excitation/emission maxima for the CellROX Green Reagent are 508/525 nm.
I need a formaldehyde-fixable reactive oxygen species detection assay. Is H2 DCFDA fixable?

H2DCFDA and similar derivatives are not fixable. The same goes for dihydroethidium and dihydrorhodamine. However, CellROX Deep Red and CellROX Green are retained for a limited time upon fixation with formaldehyde. CellROX Green may be retained upon subsequent Triton X-100 permeabilization. Avoid the use of any acetone or alcohol-based fixatives or fixatives that include alcohol, such as formalin.

Can CellROX Green dye diluted in DMSO be subjected to freeze-thaw cycles?

CellROX Green dye diluted in a DMSO stock solution is stable for multiple freeze-thaw cycles, but it is not stable long-term in aqueous solution. Please note, every time the stock solution is opened, some of the dye can oxidize, which will increase background noise. DMSO readily absorbs moisture from the air, especially when cold, so be sure to completely thaw the stock solution to room temperature before opening and only open briefly. Water in the DMSO will gradually cause the dye to precipitate and come out of solution. Minimize the number of times you use a stock solution to several freeze-thaw cycles or make small aliquots to reduce the number of freeze-thaw cycles.

What is the difference between the CellROX Green Flow Cytometry Assay Kit (Cat. No. C10492) and CellROX Green Reagent, for oxidative stress detection (Cat. No. C10444)?

The main difference between the CellROX Green Flow Cytometry Assay Kit (Cat. No. C10492) and CellROX Green Reagent, for oxidative stress detection (Cat. No. C10444), is the suggested final working concentrations and application. The CellROX Green in the CellROX Green Flow Cytometry Assay Kit is specifically designed for flow cytometry analysis and is used at a lower concentration than what is required for imaging, as fluorescence-activated cell sorting (FACS) is a very sensitive detection system.

The standalone CellROX Green Reagent (Cat. No. C10444) is marketed for fluorescent microscopy and require working concentrations appropriate for imaging. This can typically be as much as 10-fold higher than the concentration recommended for flowcytometry. The standalone CellROX Green Reagent can also be used for flowcytometry; however, the working concentration will have to be optimized. For the flow cytometry quick reference, use the link below.

CellROX Flow Cytometry Assay Kit Quick Reference
What dyes can I use to detect reactive oxygen species (ROS) in my bacteria?

Many dyes that are used on mammalian cells have also been shown to be useful in bacterial cells. For example, CellROX Deep Red Reagent has been shown to work in B. subtilis (see Reference: http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf). If you are interested in a particular dye, but are not sure if it will work on your bacteria, literature searches are the best way to check to see if it has been tested. If not, then it may be worth testing yourself.

What cellular processes can be analyzed with a flow cytometer?

-Calcium flux: Each of the Oregon Green calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca2+ and increase their fluorescence intensity 14-fold with increasing Ca2+ concentration. The cell-permeant formulation (Cat. No. O6807) can be loaded in cell media and is compatible with flow cytometry.
-Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing. Rhod-2, AM (Cat. No. R1245MP), in particular, localizes to mitochondria and can be used with flow cytometry.
-Membrane potential: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of products specifically designed to assay mitochondrial membrane potential in live cells by flow cytometry, with minimal disruption of cellular function. The MitoProbe family of mitochondrial stains (Cat. Nos. M34150, M34151, and M34152) provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis. MitoTracker dyes (Cat. Nos. M7510 and M7512) are membrane potential-dependent probes for staining mitochondria in live cells. The staining pattern of MitoTracker dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. The Mitochondrial Permeability Transition Pore Assay (Cat. No. M34153) provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. The mitochondrial permeability transition pore (MPTP) is a non-specific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death.
-Phagocytosis: In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles indicators—bacteria and yeast labeled with fluorescent dyes.
-Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used to monitor stages in the pathway. We have no-wash assays labeled with pHrodo Red or Green (Cat. Nos. A10010, P35361, P35364, P35365, P35366, and P35367) and no-wash assays for whole blood (Cat. Nos. A10025, A10026, P35381, and P35382), all suitable for flow cytometry.
-pH changes: Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. pHrodo dyes (Cat. Nos. P35373 and P35372) provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths. Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes (Cat. Nos. P35368 and P10361) provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes, with no quench or wash required.
-Reactive oxygen species: Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are non-fluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green (Cat. No. C10492), CellROX Orange (Cat. No. C10493), and CellROX Deep Red (Cat. No. C10491) Assay Kits validated for flow cytometry.

For Research Use Only. Not for use in diagnostic procedures.