Good morning to everyone,
To induce my cells, I use a medium (DMEM, 4.5 g/mL glucose) supplemented with vitamin D, vitamin C and Beta- glycerophosphate.
I used this mix for long, so I know it works fine.
Yesterday, I added vitamin D to the mix and I had an unusual reaction: the bubbles on the surface burst and a strange veil, like a milk skin, appeared and didn't dissolve.
I used the same tube of vitamin D in the previous day, and I've never had problems.
I changed medium and tube of vitamin D, but the problem presented itself again (I added vitamin before the other compounds).
Furthermore, I tried to add vitamin D in BSA- free medium, but I had the same reaction. The vitamin was reconstituted in sterile, filtered EtOH and always kept at -20.
Did someone have the same problem and could suggest me the reason?
Thank you in advance
How long were your Vit D aliquots stored? More than two years at -20C? Is it possible you witnessed the loss of solubility after long term storage? Is it possible that the ethanol was partially evaporated from the aliquot, increasing the concentration of the stock solution?
https://www.caymanchem.com/pdfs/9000683.pdf
How much of your stock solution do you use each time to make up your induction medium? What is the size of your storage aliquots? You might want to increase number of aliquots for storage when you make up your stock solution. We found that repeated freeze/thaw from same aliqout would decrease its activity, with each successive freeze/thaw. Therefore, our aliquots were made up for single use.
Vitamin D is a fat-soluble vitamin, it will bind to BSA as a carrier protein and be more stable (if you use BSA). The media should also be warm prior to addition. It all depends on the concentration, I had this issue when trying to make a higher concentration stock to dilute.
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