Dear All,
I am planning to study the effect of melatonin or H2O2 on different treating time periods to the cells, for example, treating at t0 (starting time) and t12 (after 12hr), then collect all samples at t24 (after 24hr).
There two treating solutions: (1) melatonin solution (soluble in ethanol then diluted with complete growth media) and (2) H2O2 solution (diluted with complete growth media), and then store them one or two days at 4 degree C. before treating to adherent cells such as SH-SY5Y. To prepare melatonin and H2O2, a day or two before instead of prepare fresh, could help me reduce workload on the treating day.
My questions are:
(1) Is it ok to prepare melatonin or H2O2 a day or few day, instead of fresh to treat to cells?
(2) If I prepare melatonin or H2O2 stock solution fresh and treat to cells at t0, Can I keep this stock solution at 4degreeC and use it again to treat to cells at t12? Or I should prepare another fresh solution with the same concentration at t12?
Thank you so much
Best regards,
I do not know at all, but my general experience with biologically active substances is: as an example; one of my former students lost a year in a different lab with not carefully keeping active proteins permanently on ice before using them and always using fresh material, i.e. thawing only once from an aliquoted stock solution and not refreezing it.
I do not know at all if you could, perhaps, aliquot and freeze your samples, but I would not risk wasting a year and then realize the H2O2 solution should be freshly prepared each time. Maybe you do not need to do that and others can convince you to do it the way you asked (therefore it is good to ask and discuss this) here, but my guess is, you should prepare it fresh, since H2O2 in your approach is not for preventing bacterial growth in a solution, but to always have the active substance in your cell culture - otherwise you may not be able to see protective effects if the real concentration drops in your stock solution... (but this is just my guess - maybe others really know it better and you can gain time).
I don't understand why you are planning to store melatonin with H2O2 (in the medium culture) prior to the experiment: H2O2 is highly active and is likely to induce secondary oxidizing compound when added to the culture medium, even after achieving a full protection from light exposure. Melatonin addiction, for a certain extent, may buffer the H2O2 oxidizing capacity. Indeed, a few studies demonstrated that melatonin can effectively antagonize oxidizing effects triggered by H2O2 in cells. However, let me to say it again, I reckon to be unable in grasping the rationale of that experiment. As usual, fresh solutions, prepared just before the experiment will be done, have to be preferred and I agree with Dr Eibl's suggestions. A further comment: melatonin is quite unstable in complex mixtures (ethanol + medium) and, again, I cannot recommend to prepare such solutions in advance.
best,
Mariano
I am also unfamiliar with the melatonin/H2O2 solution, but have worked with several aqueous peroxide enzymatic assays, and the peroxide solution must always be prepared fresh and kept cold until immediately before use, due to rapid decomposition of H2O2. However, convince yourself: prepare the melatonin/H2O2 solution 2 days, 1 day, and fresh (all stored 4C), and run the experiment. Sometimes the H2O2 is in such large excess that it may still be viable, despite decomposition. Personally, I would always prepare fresh peroxide solutions.
anyway, both melatonin and H2O2 solutions should be freshly prepared. You have also to consider that melatonin effects on redox balance (ROS production) are opposite when you are dealing with cancer cells: melatonin increases ROS delivery 8after h2O2 addiction) in cancer cells, meanwhile the opposite happens when non-cancerous cells are tested. Such effects have been widely discussed in the literature (you can refer to Reiter's papers).
Bests,
Mariano
Thank you Mariano,
Just one more question. In time dependent expriment, everything should keep same condition as much as possible. If I prepare either melatonin or H2O2 fresh and treat to24hr group, can I keep it for another 12hr and use it to treat to 12hr group? To prepare another melatonin or H2O2 at 12hr might cause slightly different concentration.
No. It is better to prepare both H2O2 and melatonin solutions fresh every time you need
bests,
Mariano
Hello Asa, it looks like my original "guess" and recommendation is supported by the real experts. I understand your logic that you may not want to prepare the active solutions fresh every 12 hours not only with respect to workload, but also to avoid even slight changes in concentration due to minimal variations between the different, freshly prepared solutions. Obviously the expert expect this variation much smaller than the changes due to using an older (even 12 h old) solution.
Since you may not start a test series and compare 0, 12 and 24h old solutions, you should make sure to prepare all your fresh solutions exaclty the same way (i.e. do it yourself or have the same person prepare it - otherwise there may be real changes in conditions).
Best, Robert.
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