Hi, everyone!
I am a graduate student of pharmacology from China. I am trying to measure the plasma NETs level with anti-MPO antibody and Sytox Green, which are available in our lab. Here's how I did it.
Firstly, a high-binding 96-well plate were coated overnight at 4 ℃ with anti-MPO antibody(1 μg/mL, Thermo). The plate was washed 1 time with wash buffer, then blocked with 4% BSA in PBS supplemented with 0.05% Tween-20 for 1.5 hours at room temperature. The plate was washed 3 times again, then incubate with plasm (100 μL) for 2 hours at 37 ℃, 300 rpm. The plate was washed 5 times before incubating for 15 minutes with Sytox Green in dark (100 μL, 1:1000, Thermo). The fluorescence intensity (excitation at 485 nm and emission at 535 nm) was quantified.
But there was no difference in fluorescence intensity between plasma and negative controls. I'm not sure what went wrong. I hope anybody who did it can give me some advice. Thank you so much for your generous help!
Best wished!
Yafei, Fang
There may be two explanations for this:
1) The concentration of Sytox green is too high (5mM diluted 1000 times = 5µM, which is a lot for this type of agent). It is preferable to work between 10-100nM to hope to see differences
2) Sytox green is a reagent which sticks to nucleic acid, it is impermeant to live cells. Therefore, this reagent also marks any cellular debris that is found in media, this will mask the differences between your different conditions. Flow cytometry makes it possible to overcome this signal which comes from cellular debris.
Below , please find a link for a paper describing the quantification of NETs by flow cytometry,
https://pubmed.ncbi.nlm.nih.gov/34820637/
Best regards
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