After the recombinant cytokine lyophilized powder is dissolved, repeated freeze-thaw cycles cause a severe drop in activity. What should be done?

Trehalose provides good protection in the lyophilized state, but its protection against repeated freeze-thaw cycles in liquid storage is not as direct as that of BSA. As a carrier protein, BSA can significantly reduce adsorption, but care should be taken to determine whether it affects the experimental system; for example, serum-free stem cell culture may not accept BSA. Recombinant human serum albumin or low-endotoxin BSA can be chosen. When aliquoting, it is recommended to use a high concentration and small volume, for example, aliquot 20 μL per tube at 25 μg/mL, and dilute to the working concentration when use. At -80, activity generally declines slowly for 1-3 months, but do not exceed six months. The most reliable approach is to include a newly opened tube as a control in each experiment.

Besides the diluent, aliquoting tubes and handling details are also critical. Ordinary centrifuge tubes can adsorb cytokines, so low-protein-binding tubes or siliconized tubes are recommended; low-binding pipette tips are also preferable. After reconstitution, do not wait long before aliquoting; operate on ice whenever possible. The aliquot volume should match the experiment: for example, if 10 mL of a 5 ng/mL working solution is needed each time, and the stock concentration is 25 μg/mL, then only 2 μL is required—aliquot based on this single-use amount. EGF is relatively stable, whereas FGF-2 is more prone to inactivation, so the storage conditions for the two factors should be optimized separately.

Loss of activity mainly comes from repeated freeze-thaw cycles and protein adsorption. My approach is to aliquot into single-use amounts immediately after dissolution, store at -80, take one vial and keep it at 4 degrees for use within the same day, and do not repeatedly take it out of -80 and put it back. The concentration per vial should be at least 10 μg/mL; if too dilute, it is prone to adsorption loss. Both EGF and FGF-2 are sensitive to freeze-thaw, and FGF-2 in particular also tends to aggregate, so it is recommended to swirl gently during dissolution, not vortex. If BSA-free conditions are required, low-protein-binding tubes plus trehalose or low-concentration glycerol can be used, but it is best to first perform a small-scale activity comparison.

If it has already undergone three to four freeze-thaw cycles, it is not recommended to continue using it for critical experiments, especially cell proliferation assays. The loss of activity caused by repeated freeze-thaw cycles cannot be completely reversed by adding BSA or trehalose. When reconstituting a new batch thereafter, it is recommended to aliquot the reconstituted stock solution at 5-10 μL per tube and store at -80. A final concentration of 0.1% BSA is usually sufficient; avoid high concentrations that may affect cells. Trehalose can be tested at 1-2%, but it is best to verify experimentally. For long-term storage, consider purchasing small-package powder and reconstituting it fresh as needed to avoid large-batch reconstitution.

EGF and FGF-2 lyophilized powder is not recommended to be dissolved in sterile water alone and stored long-term at -20°C. Proteins in aqueous solution tend to adsorb to tube walls, and repeated freeze-thaw cycles accelerate aggregation/inactivation. It is generally recommended to use PBS or Tris-HCl containing 0.1% BSA as the diluent; BSA can reduce nonspecific adsorption and surface denaturation. If animal-derived BSA cannot be used in cell experiments, 0.05-0.1% trehalose can be tried, but its protection against repeated freeze-thaw is limited. The aliquoting concentration should not be too low; 10-100 μg/mL is recommended. Aliquot 5-20 μL per tube based on single-use volume and freeze in low-protein-binding tubes.