2′,7′-Dichlorofluorescein diacetate (solution)

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
Using 2',7'-Dichlorofluorescein as a fluorescent probe to quantify intracellular ROS^[2]
Experimental Reagents
20,70-dichlorofluorescein diacetate (DCFH2-DA): 100 nM.
CoCl₂: 20 mM, used as an extracellular DCF fluorescence quencher.
Cell lines:
K562: Human erythromyelogenous leukemia cell line (drug-sensitive).
K562/adr: Drug-resistant subline of K562.
GLC4: Human small cell lung carcinoma cell line (drug-sensitive).
GLC4/adr: Drug-resistant subline of GLC4.
Culture medium: RPMI 1640, supplemented with 10% fetal bovine serum (FBS).
Experimental Procedure
1. Cell Culture:
(1) Seed K562, K562/adr, GLC4, and GLC4/adr cells into RPMI 1640 medium supplemented with 10% FBS, and culture in a 37°C, 5% CO2 incubator.
(2) The cell density should be 5×10⁵ cells/mL, and proceed to the next step after 24 hours.
2. ROS Measurement:
(1) Treat the cells: Add 20 μM CoCl₂ (extracellular fluorescence quencher) and 100 nM DCHF-DA, and continue incubation for 30 minutes to ensure sufficient oxidation of DCHF by ROS.
(2) Measure fluorescence: Use a Perkin-Elmer LS-50B fluorimeter, set the excitation wavelength to 502 nm, and the emission wavelength to 523 nm, to monitor the fluorescence intensity of DCF.
(3) Data analysis:
Fit the kinetic model based on fluorescence intensity changes to calculate the deacetylation rate constant (ke) of DCHF-DA and the rate constant of ROS oxidation (k2[ROSi]). Use this model to quantitatively analyze ROS concentrations in different cell lines.
Experimental Results
1. ROS concentration: ROS concentrations vary between different cell lines. The average ROS concentration in K562 cells is 0.67 nM, 0.60 nM in K562/adr cells, 0.66 nM in GLC4 cells, and 0.81 nM in GLC4/adr cells.
2. Experimental validation: The use of CoCl₂ as a quencher effectively removes external ROS signals, ensuring that the obtained DCF fluorescence signal accurately reflects intracellular ROS concentration.
Note: Drug-resistant cell lines (such as K562/adr and GLC4/adr) show higher ROS levels, suggesting that ROS accumulation may be involved in the mechanisms of drug resistance.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.