ROS H2DCFDA Reagent | ROS reagent
ROS Probes H2DCFDA
Synonyms
ROS H2DCFDA; N/A; ROS Probes H2DCFDA; ROS reagent
Label
2', 7'-dlchlorofluorescein
ExlEm
495/529 nm
Datection Method
Fluorescent
Molecular Formula
C24H16CI2O7
CAS Number
4091 -99-0
Protocols
1. Preparing the 10mM Stock Solutions Do not dissolve products until immediately prior to use. The 5 mg dyes should be reconstituted by 1 ml high quality anhydrous dimethylsulfoxide (DMSO), dimethylform-amide (DMF), or 100% ethanol.
2. Harvest the cell samples. For suspension cell line, adjust the cell concentration of the samples to 5x105 cells/ml in complete growth medium. For adherent cell line, ensure that the cells are sub-confluent. Staining of cells in phosphate buffered saline (PBS) Js not recommended.
3. Induce ROS in cells using your interested chemicals. A negative control should be prepared by incubating cells in the absence of inducing agent.
4. Prepare positive control. The cells to give a final 200um concentration of TBHP, and incubate for 30-60 minutes under normal growth conditions.
5. Dilute H2DCFDA stock solution at 1: 1 000 in prewarmed buffer (HBSS, or HEPES) to provide a final working concentration of 10uM dye. The optimal working concentration for your application must be empirically determined. Keep tightly sealed until ready to use.
6. Remove cells including negative and positive controls from growth media via centrifugation or pipetting. Resuspend cells in 1 X H2DCFDA staining solution (10 uM ).
7. Incubate at 3°C for the cells. Generally, a loading time of 10-30 minutes is sufficient.
8. Remove the loading buffer, wash cells three times with prewarmed buffer (HBSS, or HEPES); return the cells to prewarmed growth medium and incubate at 3°C for 10-20 minutes for cell ular esterases to hydrolyze the acetate groups and render the dye responsive to oxidation.
9. Observe immediately with a fluorescence microscope or a flow cytometer with 488 nm excitation using emission filter appropriate for fluorescein.
2. Harvest the cell samples. For suspension cell line, adjust the cell concentration of the samples to 5x105 cells/ml in complete growth medium. For adherent cell line, ensure that the cells are sub-confluent. Staining of cells in phosphate buffered saline (PBS) Js not recommended.
3. Induce ROS in cells using your interested chemicals. A negative control should be prepared by incubating cells in the absence of inducing agent.
4. Prepare positive control. The cells to give a final 200um concentration of TBHP, and incubate for 30-60 minutes under normal growth conditions.
5. Dilute H2DCFDA stock solution at 1: 1 000 in prewarmed buffer (HBSS, or HEPES) to provide a final working concentration of 10uM dye. The optimal working concentration for your application must be empirically determined. Keep tightly sealed until ready to use.
6. Remove cells including negative and positive controls from growth media via centrifugation or pipetting. Resuspend cells in 1 X H2DCFDA staining solution (10 uM ).
7. Incubate at 3°C for the cells. Generally, a loading time of 10-30 minutes is sufficient.
8. Remove the loading buffer, wash cells three times with prewarmed buffer (HBSS, or HEPES); return the cells to prewarmed growth medium and incubate at 3°C for 10-20 minutes for cell ular esterases to hydrolyze the acetate groups and render the dye responsive to oxidation.
9. Observe immediately with a fluorescence microscope or a flow cytometer with 488 nm excitation using emission filter appropriate for fluorescein.
Preparation and Storage
Store at -20°C for one year. Protect from light.
Shipping Conditions: Room Tempeture
Shipping Conditions: Room Tempeture
Related Product Information for ROS reagent
Introduction: H2DCFDA [2 ' ,7 ' -dichlorodihydrofluorescein diacetate] is a chemically reduced analogue of fluorescein used as an indicator for reactive oxygen species (ROS) in cells. Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2' ,7' -dichlorofluorescein.
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Product Notes
The ROS (Catalog #AAA269065) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "ROS H2DCFDA, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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