Dear all,
Thanks for answeing this question.
I am currently using the DHE to measure the ROS in adherent cancer cells. It did not work well. I put my protol in detail below. Any suggestion will be greatly appreciated.
1. Seed 250,000 cells in a 60mm dish and allow to adapt for 48 hours
2. Discard culture medium and wash with pre-warmed D-PBS for 1 time
3. Detach cells by 1ml trypsin and terminate it by adding 1ml complete culture medium
4. Centrifudge at 2000 rpm (table centrifudge) for 1 min to discard the supernatant
5. Wash the cells in phenol red-free and FBS-free HBSS
6. Centrifudge at 2000 rpm (table centrifudge) for 1 min to discard the supernatant
7. Add DHE to phenol red-free and FBS-free HBSS (final conc: 10uM)
8. Add 500ul DHE-containing HBSS to resuspend the cells
9. Incubate and culture the cells in dark in a 5ml cap-loose falcon for 30 mins at 37 degree
10. Add DMSO (control), Lapatinib (HER2 inhibitor, final conc: 500nM) and Arsenite (postive control, final conc: 100uM) and continue to culture for an additional 2 hrs
11. Centrifudge at 2000 rpm (table centrifudge) for 1 min to discard the supernatant
12. Resuspend cells in PBS on ice and keep in dark
13. Immediately do the flow analysis by excitation at 488nm (500nm also used) and PE channel for detection
I can hardly tell any difference between positivie control and DMSO. Please could anyone kindly share your experience or protocl?
Thanks in advance,
Wei
May you please elaborate your problem/issue which may directly be addressed.
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