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FerroBrite™ Green

Ferroptosis is an iron-dependent form of regulated cell death associated with the increase in lipid peroxides. Divalent iron (Fe2+) can lead to spontaneous lipid peroxidation through the Fenton reaction. Ferroptosis is regulated by signaling pathways that control iron storage and oxidative stress. FerroBrite™ Green has been developed for detecting ferroptosis via fluorescence imaging. FerroBrite™ Green has superior photostability and responds quickly to ferroptosis. The dye is permeable to live cells. Upon induction of ferroptosis, the imbalance of Fe2+ causes the reduction in the fluorescence intensity of FerroBrite™ Green. The fluorescence of FerroBrite™ Green can be readily monitored using the common FITC filter, which is equipped in most of fluorescence instruments. FerroBrite™ Green enables the real-time tracking of ferroptosis.

Example protocol

AT A GLANCE

Important Note

Before initial use, thaw FerroBrite™ Green at room temperature and briefly centrifuge to collect the dried pellet.

Subsection title
  1. Treat the cells as desired.

  2. Remove the treatment and add 100 µL of FerroBrite™ Green working solution.

  3. Incubate at 37 °C for 30 minutes.

  4. Use an Ex/Em = 450/550 filter set to monitor the fluorescence signal.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

FerroBrite™ Green Stock Solution
  1. Prepare a 5 to 10 mM FerroBrite™ Green stock solution in DMSO. For example, to make a 10 mM FerroBrite™ Green stock solution add 284 µL of DMSO to the vial of FerroBrite™ Green, and mix thoroughly.

    Note: Prepare single-use aliquots of any remaining FerroBrite™ Green stock solution. Store aliquots at ≤ -20 º C, protected from light. Avoid freeze/thaw cycles.

PREPARATION OF WORKING SOLUTION

FerroBrite™ Green Working Solution
  1. Prepare a 10 to 20 μM working solution by diluting the FerroBrite™ Green stock solution into Hanks' solution with 20 mM Hepes buffer (HHBS, AAT Cat No. 20011).

    Note: For optimal results, use this solution within 2 hours of preparation.

    Note: Protect the working solution from light by covering it with foil or placing it in the dark.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Plate the cells as desired in a 96-well black wall-clear bottom plate.

  2. Add the drug of interest to the cells at the necessary concentration.

    Note: For a positive control, add Erastin at a concentration of 10 µM to the cells. Incubate the cells with Erastin for 5 to 6 hours at 37°C in a 5% CO2 incubator to induce ferroptosis.

  3. Remove the cell culture medium and add 100 µL of FerroBrite™ Green working solution to the cells.

  4. Incubate the cells at 37°C for 20 to 30 minutes, keeping them protected from light.

    Note: The optimal concentration and incubation time for FerroBrite™ Green may vary depending on the cell line used. It is recommended to test various concentrations to determine the most effective dose.

  5. Remove the FerroBrite™ Green working solution and wash the cells twice using HHBS buffer.

  6. Add HHBS buffer and analyze the cells using a fluorescence microscope with an Ex/Em = 450/550 nm filter set.

Spectrum

References

View all 50 references: Citation Explorer
A dual-labeling fluorescent probe to track lysosomal polarity and endoplasmic reticulum dynamics during ferroptosis.
Authors: Zhao, Zhao and Jin, Wendong and Wu, Mengfan and Lin, Qingyu and Duan, Yixiang
Journal: Chemical communications (Cambridge, England) (2024): 7773-7776
Rational design of a lysosome-targeted fluorescent probe for monitoring the generation of hydroxyl radicals in ferroptosis pathways.
Authors: Zhong, Lili and Fu, Datian and Xu, Jin and Tan, Linyan and Wu, Haimei and Wang, Min
Journal: RSC advances (2024): 12864-12872
A sensitive "turn-on" Schiff-base fluorescent probe for the selective detection of Fe3+ and bio-imaging.
Authors: Che, Yiran and Yang, Jingying and Dong, Zhenming and Wang, Jianhua and Yan, Xiaoqing and Wang, Yu and Shuang, Shaomin
Journal: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy (2024): 123799
A D-π-A-type ratiometric fluorescent probe to detect polarity changes and inhibition effect during ferroptosis.
Authors: Mehmood, Abdul Hadi and Chang, Jia and Wang, Yan and Li, Shijing and Ma, Jiale and Dong, Baoli and Liu, Hong
Journal: Analytical methods : advancing methods and applications (2024): 3486-3491
Peroxynitrite imaging in ferroptosis-mediated drug-induced liver injury with a near-infrared fluorescence probe.
Authors: Liu, Ruixin and Jiang, Haijing and Yang, Wenjie and Zheng, Zhijuan and Wang, Xiaoming and Tian, Zhenhua and Wang, Danyang and Kan, Dongfang and Zhang, Dan and Tang, Zhixin
Journal: Analytica chimica acta (2024): 342673
Page updated on October 11, 2024

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Catalog Number20205
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Physical properties

Molecular weight

352.45

Solvent

DMSO

Spectral properties

Excitation (nm)

453

Emission (nm)

552

Storage, safety and handling

Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure

Platform

Fluorescence microscope

Excitation450 nm
Emission550 nm
Recommended plateBlack wall, clear bottom
Erastin induced ferroptosis in Hela cells. The fluorescence response of FerroBrite™ Green (20 µM) was evaluated in HeLa cells, in the absence and presence of 6-hour Erastin treatment at 37°C, 5% CO2 incubator. Fluorescence intensities were measured using fluorescence microscopy equipped with a FITC filter.
Erastin induced ferroptosis in Hela cells. The fluorescence response of FerroBrite™ Green (20 µM) was evaluated in HeLa cells, in the absence and presence of 6-hour Erastin treatment at 37°C, 5% CO2 incubator. Fluorescence intensities were measured using fluorescence microscopy equipped with a FITC filter.
Erastin induced ferroptosis in Hela cells. The fluorescence response of FerroBrite™ Green (20 µM) was evaluated in HeLa cells, in the absence and presence of 6-hour Erastin treatment at 37°C, 5% CO2 incubator. Fluorescence intensities were measured using fluorescence microscopy equipped with a FITC filter.