Cells were incubated with 100 M of 2',7'-dichlorofuorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany) for 30 min at 37 ºC. DCFH/DA can pass through the cell membrane and be deacetylated by cellular esterases to DCFH, which is quickly oxidized by the intracellular reactive oxygen species (ROS) present inside the cells to fluorescent DCF. To quantify intracellular ROS content, DCF fluorescence was excited at 488 nm with a 15 mW laser tuning of a FACScalibur Becton Dickinson Flow Cytometer and measured with a 530/30 filter.
Dcfh da
Manufactured by Serva Electrophoresis
Sourced in Germany
DCFH/DA is a fluorescent probe used to detect the presence and measure the levels of reactive oxygen species (ROS) in various biological systems. It is a cell-permeable compound that can be converted into a highly fluorescent molecule upon oxidation by ROS.
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6 protocols using dcfh da
1
Intracellular ROS Quantification via DCFH/DA
2
Evaluation of Oxidative Stress in Lymphocytes
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After the treatment with nanoMBGs and nanoMBG-IPs for 24 h, the oxidative stress produced in SR.D10 lymphocytes was evaluated using an analysis of the intracellular content of reactive oxygen species (ROS). Each cell sample was incubated with 100 μM 2′,7′-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany) for 30 min at 37 °C. DCF fluorescence was excited at 488 nm and registered at 525 nm with a 530/30 filter using a FACScalibur Becton Dickinson. Data acquisition and analysis were performed with CellQuest, analyzing a minimum of 104 cells in each case.
3
Intracellular ROS Quantification by DCFH/DA
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Cell suspensions were incubated for 30 min at 37 °C with 100 μM of 2′,7′-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany). DCFH/DA can penetrate the cells and can be hydrolyzed by cytosolic esterases, producing DCFH, which is instantly oxidized by ROS to DCF, highly fluorescent and whose fluorescence intensity depends directly on the intracellular content of reactive oxygen species (ROS). DCF fluorescence was measured in a FACScalibur Becton Dickinson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. A total of 104 cells were analyzed in each sample in order to ensure a correct statistical significance.
4
Measuring Intracellular ROS Levels
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Cell suspensions were incubated with 100 μM of 2′,7′-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany) for 30 min at 37 °C. DCFH/DA penetrates into the cells, where it is hydrolyzed by cytosolic esterases and produces DCFH, which is instantly oxidized by the intracellular reactive oxygen species (ROS) to the highly fluorescent DCF. The fluorescence intensity of DCF is directly dependent on the intracellular ROS content and was measured on a FACScalibur Becton Dickinson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. To obtain statistically significant results, 104 cells were analyzed in each sample.
5
ROS Detection in Macrophages
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After exposure to the DAMs, RAW-264.7 macrophages were detached and cell suspensions were incubated with 100 μM of 2′,7′-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg, Germany) during 30 min at 37 °C. DCF fluorescence depends directly on the intracellular content of reactive oxygen species (ROS) and was measured in a FACScalibur Becton Dickinson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. In each sample, 104 cells were analyzed.
6
Intracellular ROS Measurement by DCFH/DA
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Cells were incubated at 37ºC for 30 min with 100 M 2´,7´-dichlorofluorescein diacetate (DCFH/DA, Serva, Heidelberg/Germany) for directly measuring the intracellular content of reactive oxygen species (ROS). DCFH/DA is diffused into cells and is deacetylated by cellular esterases to non-fluorescent DCFH, which is rapidly oxidized to highly fluorescent DCF by ROS. To measure the intracellular ROS content, the DCF fluorescence was excited by a 15 mW laser tuning to 488 nm and the emitted fluorescence was measured with a 530/30 band pass filter in a FACScalibur Becton Dickinson Flow Cytometer.
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