hi

you are very clever . i think use 1mM NMN final concentration  in culture medium 24 hour  for other cell lines (such as HUES7 or H9  ).

" A promising anti-ageing drug is about to undergo human trials for the first time"

good luck

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4113526/

http://www.sciencealert.com/a-promising-anti-ageing-drug-is-about-to-be-tested-on-humans

Hi. Thank you for your answer. After reading the literature I agree that a final concentration of 1mM is the usual dosage, the range I found from about 5 studies was 1uM - 10mM with 500uM-1mM being the most common. I find 1mM to be high and expensive, so I am going to use 200nM - 20uM range and see what happens. Thanks again!

Thank you for making the question as it has been useful for me as well! I just wonder if you have found the lifetime of NMN in culture because I haven't! I would really appreciate some help with this info, cheer, Fab

Hi Fabrizzio,

I am glad you got use out of it! I hope I understand your question correctly first off - I think you are asking about the kinetics of NMN activity in media? For that question I am sorry I can't be of much help because I saw no change in replicative lifespan (# of population doublings) of primary cells (healthy control and disease of interest) with NMN treatment so I did not continue working on this pilot project and it went no further. But it is a good point that maybe the drug itself was not active for long enough and different treatment regimens could have been tested. I treated cells every other day - I added drug to the media just before adding the media to cells, both media and drug were always aliquoted and warmed to room temperature just prior to adding to cells. I tested 200nM, 2uM, 20uM, and 1mM for a subset of my cells, as well as vehicle control. I did try to quantify NAD/NADH ratio using a kit from Sigma (NMN was also purchased from Sigma) based on abs450nM because NMN should be raising the concentrations of NAD/NADH nucleotides in the cultures if it is indeed fully active and working properly at sufficient concentration but unfortunately my values didn't fall within the NADH standard curve because I think the dilutions and total starting material I used were not optimal (too high and too low, respectively). I could have repeated it but I did not bother since my original experimental end point of replicative lifespan was not a positive result to begin with.

Good luck Science-ing!

Thank you very much Brenda for your answer. A shame you didn't continue but sometimes it's important to move on. Anyway the information you have provided is really valuable as looks like there is not that much experience with NMN in cell culture. If for any chance you have found or read about the NMN kinetics in culture I would really appreciate you could share this information. In terms of the concentration you mention, which was the best for you? Did you have any other measurement to prove an increase of NAD/NADH? What cell line did you use? Thanks again for your tremendous help and thanks in advance for your answer,

Kind regards,

Fab

Hi Fabrizio, you're welcome.

I did not do dosage curve or much else to test concentrations or effective dosage of NMN, just pulled information from the literature and tested multiple concentrations. In terms of replicative capacity of my cell lines, I actually saw no change from vehicle control at any concentration used in any of the cell lines I tested so I can't say which was best. I also looked at live cell mitochondrial dynamics with 10-NAO (vital dye binds to cardiolipin - mitochondrial marker) and TMRE (mitochondrial membrane potential marker) and I didn't see any strong changes but maybe a slight trend toward increased mitochondrial membrane potential at 200nm-20uM but I felt there was too much variation and I didn't have enough control. I tested human primary fibroblast cell lines with and without mitochondrial dysfunction - IMR90, and Control cells from healthy individuals compared to primary cultures from individuals with mitochondrial disease: MELAS and Friedreich's Ataxia. I don't know if this could be useful information but I diluted NMN in water, maybe a different solvent is better for activity. Again, I did not adequately control during this pilot and I was not invested in it so I didn't do great work which is why I warn you to take all I am saying here with a grain of salt :). In terms of literature about NMN kinetics, I don't know of any article but I would suggest you to look up David Sinclair's research on NMN as you might find something interesting and potentially helpful.

Best of Luck!

I hope you have read the attached info.

Dear Colleagues, I have much more complicated task: I need to give the NMN to a human. (First, I will test it on myself.)

Have no idea about dosage, half-life in human body, penetrating the blood-brain barrier, stability in digestion, way of administering: oral, sublingual or what?

Would be very grateful for your any hints on this matter