Life Technologies
CM-H2DCFDA (General Oxidative Stress Indicator)
C6827
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Reactive oxygen species (ROS) include oxygen ions and peroxides. When the cell is stressed, it can result in significant damage to cell structures. Upon treatment with novel anti-cancer drugs, it is useful to assess the level of ROS generation in the cancer cells. We needed a ROS-detecting system that would work in flow cytometry assays. This product was recommended by a colleague.
Flow cytometry
Cells, control and treated
1. Grow cells to about 70% confluency before treatment 2. When you are ready to measure ROS, trypsinize cells or use a scraper and wash them in PBS. 3. Cells are then counted on the hemocytometer to have the same amount of cells stained per sample 4. Transfer the cells to a labeled 5ml polystyrene round bottomed tubes. 5. Remember to always have an unstained control (PBS only) 6. Dilute DCF in PBS (at this point avoid any direct light exposure to the probes. Turn off the light above and in the hood.) 7. Add the probe to the cells and incubate at 37C for 30-45mins. 8. Remember to always have an unstained control 9. After incubation, add an extra 1 ml of PBS and spin down cells. 10. Resuspend cells in 200µl of PBS and ensure no cell clumps present 11. Remember to always have an unstained control (PBS only) 12. Optionally you can have a H2O2 control in which you added 100µM H2O2 to cells 0.5-1hr prior to cell harvest.
Preoptimize the dilution for your cells!
As shown in the graph, no DCF staining results in no fluorescence in the FL-1 (FITC) channel. Treatment with etoposide leads to an increase in ROS generation compared to control cells (no treatment).
Use media fluorescence intensity to compare populations (rather than percentages)
Strong staining
On the expensive side
This is a good, reliable probe. It stains well, and is easily detected by most flow cytometry machines.
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