I have aliquot the same product from Sigma (D1530) in 100% ethanol previously at a concentration of 100 microM concentration and stored at -20 degree. But I am not finding any activity after some time. I am performing MTT and expecting to have some toxic effect on them as mentioned in many literature. I even got cytotoxicity for the first time, when I freshly aliquoted. But, after that I am unable to get the same result not even near to it.
The compound is very unstable in aqueous solution. The best is to extemporaneously prepare it. If you want to store for short duration, try storage at -80°C, but it completely disappears after 6 months.
I will confirm by HPLC to make sure all compound is gone. If you do not have a method, Lab which runs 1,25- dihydroxy Vitamin D3 method in the hospital can easily test.
Vitamin D metabolites are unstable in solution, even at -20ºC. My suggestion is always prepare solutions always from solid compounds, that are quite stable at -20ºC. You may extend the lifetime of your stock solution (ca 10-3 M) storing it at -80ºC. Working solutions (ca 10-5 M) should always be checked by UV-VIS and do not last longer than a month.
For short time storage use high concentration ethanol (methanol) solution and story it in -80 (watch out for precipitation). You have to check concentration every time you use it. The simplest method is to use nanodrop or similar equipment. But you have to scan 220-320 nm in order to draw characteristic spectrum for vit D (If you observe any alternation in UV spectrum vitamin D is no good). A maximal UV absorption at 265 nm with a molar extinction coefficient of 18,200 for both vitamin D2 and D3. I have just noticed that some papers use 254 and 18.300. Of course HPLC is much better. Also you can make aliquots and dry them off (10 ug-100 ug a vial) then store in -80.
10-2 M in 100% Ethanol and store at -80°C. It's stable for at least a year.
We have stored it in ethanol at -80C and tubes wrapped in foil as it is light sensitive.
Prepare a concentrated stock solution in ethanol and keep it at -20º/-70º
When adding it to cell culture, make sure to keep final ethanol concentration below 0.1%
I agree with Jose A. Riancho. I t lasts for a long time as a concentrated stock solution even at -20.
Prepare a stock solution 10-3M in 100% Ethanol and replace the air in the vial with a flow of Nitrogen. Store the vials at -80 °C and prepare a fresh working solution every time you need. Work always without a direct light and wrap the vials with foil.
1 mM in ethanol and keep it at -80 degree. It is stable for at least 1 year.
Dissolve in absolute alcohol to make conc. of 100mM.Store at -80 degree.
As above and the added benefit of storing under argon. Spray argon on top before putting into -80C will help stop oxidation.
You should work in a dry box in order to avoid oxidation and, consequently, loss of bioactivity. You should also aliquot it in the dark (or under red light and you should protect the aliquots, from the light , while you're working with cells. the storage must be at -80°C always protected from the light. You could grossly monitor the residual activity of vitamin D, over the time, spectrophotometrically (there are a number of papers on that , published long time ago).
Thanks to all for guidance.
Should I use absolute alcohol or 95% alcohol as given in product data sheet and stored at -20 degree freezer.
previously I have made 100 microMolar stock and alliquoted 0.1mL in 0.5ml oppendorf protected from light as well.
agree 95% and absolutely keep it in the dark, i always have lights off and wrap tubes in foil, especially when making up media.
Thanks Anna and Miguel.
Anna will you please let me know what is the concentration of your main Stock. And at what temperature have you stored you Master and working stock.
Our concentration of your main stock is aprox. 5 10-2M, we stored it a -80ºC in and amber vial and we work with aprox. 10-5_10-6M solutions in amber vials kept at -20ºC and they last at least for two weeks.
Thanks Miguel Maestro
You mean to say 10-5 to 10-6M of Vit D3 in culture media.
I am planing to make 5X 10-4M in 95% ethanol in amber Vials and will store at -80.
main stock is stored at -80C @ 10mM (10^-2) and working stock made to 100uM (10^-4) and I use it at 1000-10nM (10^-6-8).
Hello Anna,
Will you please tell me which medium/ solvent you use for preparation of working stock from master stock?
And about conditions of storage and stability.
Can I preserve the aliquot in nitrogen vapour to achieve low temperature and protect from oxidation.
hello,
Good suggestions for the storage of 1, 25- dihydroxy vitamin D. I am having cholecalciferol. How can I store it in the form of aliquots in 95% ethanol? Please help me in this respect, as my old stock got oxidized...
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