How to determine whether the flocculent precipitation of thawed fetal bovine serum is normal or spoiled?

First, observe the precipitate characteristics: white flocculent, does not disperse upon shaking. In fetal bovine serum, this is often fibrin or lipoprotein precipitation, especially more pronounced during rapid thawing at 37°C or repeated freeze-thaw cycles. You can centrifuge at low speed, 3000 g for 10 minutes, and check whether the precipitate is loose and the supernatant is clear; if the supernatant is clear and the precipitate volume is small, it is most likely normal precipitation. The fact that previous batches had less does not indicate an abnormality; different serum batches vary in their tendency to precipitate. It is recommended that in the future, after removing from -20°C, thaw slowly at 4°C, and after thawing, mix gently before aliquoting. Do not place directly in a 37°C water bath, as this will reduce flocculent precipitation.

SDS-PAGE can be performed, but it is best to run the same batch of normal serum and the problematic batch side by side on the same gel to see whether the protein bands show obvious degradation or loss of the main band; for samples with flocculent precipitate, centrifuge first, then take the supernatant and precipitate separately for gel comparison. If there is no difference in the bands, the precipitate is very likely fibrinogen-type precipitation. Considering that one 500 mL bottle costs over three thousand, it is recommended that you take photos, record the batch number and precipitation conditions, first communicate with the supplier in writing, and keep the unopened bottle; if abnormalities appear in cell function validation or mycoplasma testing, then proceed with return or exchange. Generally, normal precipitation can still be used after centrifugation or filtration, but if you are not reassured, requesting a replacement is reasonable.

Flocculent precipitate cannot be directly used as evidence of mycoplasma contamination, as mycoplasma is typically invisible to the naked eye. You can take 1 mL of serum for PCR mycoplasma testing, and simultaneously filter the remaining serum through a 0.22 μm filter. Culture RAW264.7 cells with both unfiltered and filtered serum, and observe cell morphology, proliferation, and adherence; if poor cell condition or signs of contamination still occur after filtration, greater attention should be paid to other issues. Filtration itself will lose some macromolecules, but it does not affect short-term assessment. If the precipitate disappears after filtration and the cells are in good condition, it indicates that the precipitate is more likely due to physical precipitation.

Same question here. After thawing, I also noticed a small amount of flocculent material. Can it be used after centrifugation?

Thawing overnight at 4°C will result in much less loss; do not use rapid shaking at 37°C.