How long can fetal bovine serum be stored at 4 ℃ after thawing? How to store and prevent contamination after opening?

After the entire bottle of serum has thawed at 4°C, I generally recommend using it within 1-2 weeks, with a maximum of about 2 weeks. This is because 4°C is only refrigeration, and some proteases and complement components will still change slowly; the longer the time, the more likely flocculent precipitate and pH drift will occur. If you cannot use it up in the short term, it is best to aseptically aliquot it while it has just thawed and is still cold, dividing it according to the amount used per experiment, such as 50 ml or smaller, and then refreeze it at -20°C. Once the entire bottle has been thawed and then refrozen, it counts as one freeze-thaw cycle for the serum, and each subsequent aliquot thawing counts as another, so try to limit the total number of freeze-thaw cycles.

Adding dual antibiotics directly to a whole bottle of serum is not recommended. Dual antibiotics can mask potential contamination, and not all cells tolerate long-term high-concentration antibiotics; if you are concerned about contamination during handling, you should add them according to the instructions when preparing complete medium, rather than adding them to the serum bottle. The key to contamination prevention is biosafety cabinet UV and ventilation before aliquoting, wiping the bottle mouth with 75% alcohol, using disposable pipettes or an electric pipette, sealing tubes tightly with parafilm, and keeping the bottle mouth above the water during water bath thawing. After aliquoting, freeze each tube separately to avoid repeated bottle opening.

Since there is no -80°C freezer, and thawing the entire bottle at 4°C means it cannot be used up in one go, the most practical approach is "thaw once, aliquot once, use one aliquot at a time." Once thawed, the whole bottle should preferably not be stored long-term at 4°C as a working solution, because daily retrieval will repeatedly introduce air and opportunities for contamination. It is recommended to aliquot into small portions as soon as possible today and store them frozen at -20°C; thereafter, thaw only one aliquot at a time. If this batch has already been stored for more than 3 days, it is advisable to first centrifuge at low speed to check for precipitate, and to perform a sterility test when preparing complete medium (take a small amount and place it in a 37°C incubator overnight to check for turbidity). Before critical experiments, first test growth with one cell line.