Dihydrorhodamine 123 (DHR 123) (Synonyms: DHR 123)

This plan only provides a guide, please modify it to meet your specific needs.

1.Solution Preparation:

(1) Preparation of stock solution: Allow the solid stored at low temperature to equilibrate to room temperature. Dissolve Dihydrorhodamine 123 in DMSO to prepare a stock solution with a concentration of 5 mM.

Note: Unused stock solutions should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freeze-thaw cycles.

(2) Preparation of working solution: Before the formal experiment, dilute the stock solution to the desired working concentration using an appropriate buffer (e.g., serum-free medium or PBS).

Note: The optimal working concentration should be adjusted based on the actual situation or determined by consulting the literature and setting up a gradient concentration experiment. The working solution must be prepared fresh and used immediately.

Using DHR123 to Detect Intracellular ROS Steps ^[1] (from literature, for reference only)

(1) Cell treatment: Wash the cells with water to remove any contaminants or debris.

(2) DHR123 incubation: Resuspend the cells in PBS containing DHR123 (15 μg/ml, approximately 43μM). Incubate with shaking at 30°C for 90 minutes.

(3) Washing cells: Wash the cell pellet twice with PBS buffer to remove excess DHR123.

(4) Fluorescence detection: Measure the fluorescence of 10,000 cells using a flow cytometer. Set the excitation and emission wavelengths to 485 nm and 535 nm, respectively, to detect the fluorescence emitted by the reaction of DHR123 with intracellular ROS. Analyze the fluorescence distribution in the samples using FlowJo 7.6.5 software.

Using DHR123 to Detect Intracellular ROS Steps ^[2] (from literature, for reference only)

(1) Cell treatment: Treat cells according to experimental requirements.

(2) DHR123 incubation: Prepare a solution containing 2.5 μM DHR123 in HBSS buffer solution. Incubate the cells with DHR123 solution at 37°C for 30 minutes.

(3) Washing cells: Wash the cells twice with HBSS to remove excess DHR123.

(4) Lysing cells: Lyse the cells with RIPA buffer to release the intracellularly converted Rh123⁺.

(5) Fluorescence detection: Set the excitation and emission wavelengths to 485 nm and 535 nm, respectively, to detect Rh123⁺ fluorescence. Record the fluorescence intensity readings as relative units indicating intracellular ROS levels.

Precautions: This experimental protocol provides guidance for detecting intracellular reactive oxygen species (ROS) using Dihydrorhodamine 123 (DHR123) and subsequent fluorescence measurement. Adjustments can be made based on specific experimental requirements.

References:

[1]Antoine Simoneau, Étienne Ricard, Sandra Weber, et al. Chromosome-wide histone deacetylation by sirtuins prevents hyperactivation of DNA damage-induced signaling upon replicative stress [J] Nucleic Acids Research, Volume 44, Issue 6, 7 April 2016, Pages 2706–2726.

[2] Wing-Kee Lee, Stephanie Probst, Bettina Scharner, et al. Distinct concentration-dependent oxidative stress profiles by cadmium in a rat kidney proximal tubule cell line[J] Archives of Toxicology. 30 January 2024.