Because I would like to know the sperm DNA lysis buffer along with NBT action in overnight incubation. Is there any detrimental effects on sperm cell membrane? Relates to apoptosis and ROS of membrane? Because NBT is used to measure ROS in sperm cells easily as well as quickly.
Article Investigation on the Origin of Sperm DNA Fragmentation: Role...
We have data about both increase of PS expression and increased DNA damage after overnight incubation
It is not clear from your question what you really want to do. However, following text may be useful for your experiments:
If you want to measure NBT reduction by superoxide anion, you should incubate sperm cells with NBT in an adequately aerated buffer isotonic to sperm cell. Later on you can quantify amount of formazan formed in the incubation buffer.
If your interest is to measure ROS generation by lysed sperm cell membrane, you should also include NADPH along with NBT. Later on you can quantify formazan as well as DNA damage.
For intracellular ROS measurement you can use fluorescent dyes such as H2DCF-DA and DHE.
Is there a cheap method to measure ROS accumulation or production in the sperm using a simple optical microscope without fluorescent dyes or chemiluminescence?
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