After thawing fetal bovine serum, if white flocculent precipitate appears, can it still be used?

Aliquoting and freeze-thaw cycles are critical. After the serum arrives, it is best to thaw it at 4°C and aliquot it immediately. Mix gently and aliquot into 50 mL/tube or 15 mL/tube. Store short-term at -20°C and long-term at -80°C. Each time, take one tube and thaw it at 4°C overnight. Do not thaw quickly in a 37°C water bath, and do not repeatedly freeze-thaw the entire bottle. Repeated freeze-thaw cycles will aggravate protein precipitation and reduce active components. During aliquoting, pay attention to aseptic technique. Use disposable pipettes or a bottle-top dispenser, and wipe the tubes and bottle mouth with 70% alcohol. Many precipitation problems are actually related to repeated freeze-thaw cycles.

For primary neurons, serum batch-to-batch variation may affect the results more than the precipitate itself. It is recommended to perform a small-scale pilot experiment first when a new batch arrives, focusing on neuron viability, neurite length, glial cell proportion, and even immunofluorescence markers. Do not wait until the formal experiment to find out. If the cell condition fluctuates after precipitate treatment with this serum batch, it is not recommended to continue using it; find a replacement from the manufacturer or another brand as soon as possible. Alternatively, you can lock in a validated batch, stock up enough in advance, and reduce mid-experiment batch changes.

Whether to ask the manufacturer for a replacement depends on the batch situation. If there is obvious flocculation in the stock solution right after opening, it does not disperse upon shaking, and the volume is not low, it is advisable to take photos, retain the original bottle and lot number, and contact the distributor. Some manufacturers may consider a small amount of fibrin precipitation to be normal and may not handle it as a quality issue; however, if the cell condition is indeed poor after filtration, you can use the data to make your case and request a different lot or a return. Generally, I would first ask the distributor to send a trial sample or a small sample from another lot. For primary cell experiments, do not wait three weeks, as it will affect your progress.

During processing, you can let it stand at 4°C for several hours to allow the precipitate to settle, then gently aspirate the supernatant with a sterile pipette, or centrifuge at 4°C, 500–1000g for 10 minutes. If filtration is necessary, it is recommended to use a 0.45 μm low-protein-binding filter membrane; do not directly use a 0.22 μm filter, as it clogs easily and causes high shear stress. Filtration will result in the loss of some large-molecule attachment factors, with little effect on tumor cells but possible variability for primary neurons. Therefore, first take a small amount of the filtered solution to test neuronal survival and neurite outgrowth, and once confirmed to be fine, use it for formal experiments.

White flocculent precipitate is, in most cases, fibrinogen or lipoprotein that precipitates out during low-temperature thawing, and is especially common after overnight thawing at 4°C; it is not necessarily microbial contamination. If the flocculent material is scant and the supernatant is not turbid, it can generally be handled by low-speed centrifugation or by standing and collecting the supernatant. However, primary neurons are very sensitive to serum quality, so I suggest not simply filtering it and proceeding; first run a small-bottle test. I have encountered something similar before: after filtration, neuronal attachment worsened, and later it improved after switching lots. If the amount of sediment is large or the entire bottle is hazy and turbid, then you should be on alert.