HAPI cells and microglia were planted in a 12-well plate and then treated with varying concentrations of the test compound or drug. After 12 h, cells were incubated at a final concentration of 5 µM DCFH-DA (WLA131, wanleibio, Dalian, China) for 30 min at 37°C, after which they were washed, dyed with DAPI, and immediately analyzed for fluorescence intensity under a laser confocal microscope with a ×20 objective lens.
Dcfh da
Manufactured by Wanlei
Sourced in China
DCFH-DA is a fluorescent probe used for the detection of reactive oxygen species (ROS) in biological systems. It serves as a cell-permeable indicator for oxidative stress and can measure intracellular levels of ROS such as hydrogen peroxide, peroxynitrite, and hydroxyl radicals.
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4 protocols using dcfh da
1
Cellular Oxidative Stress Assay
2
Intracellular ROS Levels Analysis
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2,7-Dichlorofluorescein diacetate (DCFH-DA) (#WLA131; Wanlei Bio) was used to
test the intracellular levels of ROS, which included hydroxyl free radicals
(radical · OH), hydrogen peroxide (H2O2), and superoxide
anions (O2·−). CoQ10 was administered to cells with or
without 10-4 M H2O2 and treated with 50 ng/mL
RANKL. Afterwards, 10 μM DCFH-DA was added to each well, and the mixture was
incubated in the dark for 15 min at 37°C. A multifunctional microplate analyzer
(Tecan, Infinite M200 Pro, Switzerland) was used to measure the fluorescence
values.
test the intracellular levels of ROS, which included hydroxyl free radicals
(radical · OH), hydrogen peroxide (H2O2), and superoxide
anions (O2·−). CoQ10 was administered to cells with or
without 10-4 M H2O2 and treated with 50 ng/mL
RANKL. Afterwards, 10 μM DCFH-DA was added to each well, and the mixture was
incubated in the dark for 15 min at 37°C. A multifunctional microplate analyzer
(Tecan, Infinite M200 Pro, Switzerland) was used to measure the fluorescence
values.
3
Intracellular Reactive Oxygen Measurement
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The cells were inoculated in 6-well plate medium. Cells were incubated overnight before being treated with CAP. After treatment, the cells were continued to be cultured in a cell incubator at 37 °C and 5% CO2 saturated humidity for 24 h. After reaching the time, each group of cells was collected and washed twice with PBS. The cells were incubated in an incubator at 37 °C for 20 min, inverting and mixing every 5 min, with 1 mL of DCFH-DA (Wanleibio, China, WLA131) dilution (1:1000 in medium). Cells were washed 3 times with PBS to fully remove any DCFH-DA that had not entered the cells. 500 μL of PBS was used to resuspend the cells, and then flow cytometry (Aceabio, USA, NovoCyte) was used for flow detection.19 (link)
4
Oxidative Stress Markers in Spinal Cord Injury
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Iron accumulation in SCI was tested using the tissue iron assay kit (A039-2-1, Nanjing Jiancheng Bioengineering Institute). Malondialdehyde (MDA), as a natural product of lipid peroxidation, was quanti ed using Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime). The protocols of iron and MDA concentration tests were performed according to the menufacturer's instructions and the absorbance was measured at 532 nm and 520 nm respectively. ROS assay ROS production was determined by chemiluninescence using the uorescent probe 2′, 7′-dichlorodihydro uorescein diacetate (DCFH-DA, WanleiBio). After anesthetization, rats were perfused transcardially with precooled PBS and the spinal cords were digested by pancreatin to obtain single-cell suspensions. Cells were incubated with DCFH-DA (10 μM) at 37℃ for 20 minutes. Later, the cells were observed and pictured via the inverted uorescence microscope (Axio Observer 3, Germany). The ROS uorescence intensity was calculated by software Image J.
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