How to determine if there is hemolysis or high endotoxin levels in the new batch of fetal bovine serum?

First, perform centrifugation and observation: take 1 mL of thawed serum and centrifuge at 3000 g for 10 min to check whether the upper layer is clear and whether there are red blood cell fragments in the lower layer. If the supernatant is obviously red or has absorption peaks at 414 nm and 560 nm, it strongly suggests hemolysis; measuring the absorbance at 414 nm with a spectrophotometer and comparing it with the previous lot is more objective. A slightly darker color is not necessarily hemolysis; fetal bovine serum itself varies in color between lots. It is recommended that you retain 1-2 mL of the old lot for parallel comparison to avoid misjudgment based on color alone.

The sample size is not large and the cells are mesenchymal stem cells. Personally, I suggest not drawing conclusions based solely on self-testing. You can retain one unopened original vial and one aliquot of the thawed sample, and send them to a qualified third-party laboratory for endotoxin, hemoglobin, and basic biochemical testing, which is usually more convincing than self-testing results. At the same time, request the CoA and factory endotoxin value for that lot from the supplier, and combine these with your adherence results once they are near the normal range. If there is a clear difference in adherence between the old and new lots, even if the test values show no obvious abnormality, you should still request a lot replacement or return, because cell culture ultimately depends on functional performance.

For endotoxin testing, you can start with the domestic limulus amebocyte lysate gel-clot method. A kit with a sensitivity of 0.25 EU/mL is sufficient for small samples, but be aware that proteins and salts in serum may interfere. Dilution and positive controls must be performed according to the instructions, otherwise false negatives are likely. Rapid detection kits can be used for initial screening but cannot replace formal quality control. Mesenchymal stem cells are highly sensitive to batch-to-batch differences, and poor adherence may also result from fluctuations in growth factors between batches, not necessarily endotoxin. It is recommended to simultaneously perform a simple adherence rate experiment: plate the same batch of cells with old and new serum separately, and observe the number of adherent cells at 6 hours and 24 hours.

A dark red color does not necessarily indicate hemolysis, but caution is warranted if the hemoglobin level exceeds 30 mg/dL.

High endotoxin levels indicate poor cell condition. It is best to send each batch for testing or have the manufacturer provide a report.