I am trying to use the invitrogen CM-H2DCFDA for ROS detection. This is the first time Im trying this assay. I would be really grateful if anyone else who has experience with this assay help to answer these questions.
1. Is it all right to store the CM-H2DCFDA at -20oC ? I have 25mg vial and measure out some powder each time I will be doing the assay. Is this ok ? The product sheet gives some strict conditions. But Im not sure how to stock the reagent. is there any precautions to take when handling the reagent to prevent it from losing activity when measuring out powder.
2. I want to treat suspension cells with various stimulants for over long periods of time 2 hours, 4 hours, 6 hours, 12 hours etc. and then measure ROS. I tried to stimulate and then load the cells with the dye and detect fluorescence but it failed. I cant see a difference between H2O2 stimulated cells and non stimulated cells measured. I used Log FL1 is this appropriate ?
3. After various time points in stimulation I centrifuged the cells and added the probe in PBS for 30 minutes at 37oC. the concentration was 10uM final conc. Is it allright to centrifuge the cells and wash cells after loading the probe ?
I do hope some one can help me,
ROS detection using CM-H2DCFDA
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