What should we do if the serum thawing time is too long and we urgently need to prepare the complete culture medium?

In an emergency, a room-temperature water bath may be used, but do not use 37°C. Place the serum bottle into a clean sealed bag or wrap the bottle opening, then place it into a 20-25°C water bath and gently swirl once every 10-15 minutes. A full 500 mL bottle typically takes several hours to thaw completely, depending on the water bath volume and initial temperature; it is not advisable to promise a fastest time. When only a small amount of ice crystals remains, mix gently; after it is completely thawed, proceed with solution preparation. A room-temperature water bath provides more uniform thawing than shaking by hand. Localized heating from the palm can easily affect serum stability and also easily lead to contamination.

It is correct to aliquot uniformly at 50 mL. It is recommended to thaw completely at 4°C once, mix gently, aliquot into 50 mL portions in a biosafety cabinet, and store at -20°C or -80°C, labeling with lot number and date. Although one additional freeze-thaw cycle of serum is not ideal, many laboratories accept it in emergencies; do not repeatedly freeze and thaw. Whether to buy 100 mL or 500 mL depends on monthly usage: if 500 mL cannot be used up within 4-8 weeks, 100 mL is more reliable; if it can be consumed within 2-4 weeks, 500 mL aliquoted is more economical. For CCK-8 experiments, it is best to use a fixed serum lot number.

If you plan to seed the plate for CCK-8 tomorrow, do not hastily thaw it at high temperature. You can quickly check whether there is still aliquoted stock available, or borrow a small amount of serum from the same source for a control; if it can only be handled on site, a room-temperature water bath with frequent swirling is a relatively acceptable short-term option. After it is completely thawed, if obvious precipitate is seen, you can centrifuge at low speed and take the supernatant; filtration is not mandatory. More importantly, in the future keep one 50 mL tube thawed at 4°C and use it up within a short period, so as to avoid being pressed for thawing time again next time.