Hello, I am interested to perform ROS measurement from isolated rat's sperm by flow cytometry by H2DCFDA. So, is there any standard protocol which I need to follow to get the results along with sample preparation and interpretation of the data from the images which I get. Any help or suggestion regarding this matter is highly appreciated. Thank You.
Hello Sagnik.
You could follow the procedure at the links.
Article Reactive Oxygen Species (ROS) in human semen: determination ...
https://www.researchgate.net/post/Dihydrorhodamine_123_for_ROS_measurements_in_vitro
If you use propidium iodide together, you could get more precise value by eliminating the dying, dead sperm with DC-FDA.
However, both dyes are toxic to cells, you need to pay attention to minimum concentrations of them. In particular, the solvents dissolving the both dyes are harmful to living cells under prolonged incubation with dyes.
Best wishes.
Hello Sagnik.
You could follow the procedure at the links.
Article Reactive Oxygen Species (ROS) in human semen: determination ...
https://www.researchgate.net/post/Dihydrorhodamine_123_for_ROS_measurements_in_vitro
If you use propidium iodide together, you could get more precise value by eliminating the dying, dead sperm with DC-FDA.
However, both dyes are toxic to cells, you need to pay attention to minimum concentrations of them. In particular, the solvents dissolving the both dyes are harmful to living cells under prolonged incubation with dyes.
Best wishes.
Thank you so much Sir for your guidance regarding this matter. Yes Sir I have propidium iodide. The stock solution of DCFDA is 5 millimolar concentration in DMSO but I am confused regarding the concentration of working solution. 100 microlitre cell suspension is taken and 400 microlitre PBS is added, now how much PI and DCFDA should I add and incubation time is also a factor regarding this matter. Moreover, I would also like to use JC 1 dye and Apoptosis detection kit Annexin V FITC with PI on the same sample. Any help or suggestion regarding this matter is highly appreciated. Thank You.
HI, the intracellular levels of ROS can be measured by flow cytometry using dihydrofluorescein diacetate (DCFH) to detect intracellular hydrogen peroxide radicals. This dye is oxidized to the highly fluorescent derivative dichlorofluores . Read the document attached here, it will elp you more.Goodluck.
Hi Sagnik.
Annexin V-FITC kit (including PI) may be complicated with JC-1 dye under co-staining due to the their wave lengths and three fluorescent dyes. Make sure that they are separable under cytometry by studying their emission wave lengths. All dyes are OK if each of them is used alone, combined use of two more dyes would cause cell damage, overestimating dying or dead cells cells, and also would limit the interval premitted during your staining and washing procedures.
Regards.
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