Technical Information
Atlas Biologicals firmly believes that no questions should go unanswered. We want you, as the end user, to be educated on all aspects of Fetal Bovine Serum.
To help provide a better understanding of technical terms and standard procedures please read below. If you have any further questions or need any clarification please contact us with any inquires.
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Thawing Frozen Serum
There are several methods used to thaw serum, however, the most important aspect of thawing frozen serum is that it must be mixed or swirled during thawing and most definitely after it is complete.
Thawing at 2°C - 8°C in a refrigerator or at room temperature
If serum is thawed at room temperature or in a refrigerator with no mixing, a color gradient will form in the bottle going from light at the top to dark at the bottom. This is the result of lipids, proteins and salts settling out and concentrating at the bottom of the bottle which can lead to the formation of crystalline or flocculent precipitates. These precipitates are not toxic to cell cultures, but they affect the appearance and consistency of each bottle of serum. Small amounts of precipitates are not uncommon, even in serum that is thawed using proper recommended procedures. This is normal and will not affect product performance. If a bottle is left to thaw without mixing and a gradient forms, when it is gently swirled, you will see sheer lines in the serum as the contents mix and return to equilibrium. When the sheer lines are no longer visible and the serum is clear the contents are uniform.
If thawed serum is allowed to stand for long periods of time, greater amounts of precipitates will form and are often insoluble. Filtering serum to remove precipitates is not recommended and could result in the loss of nutrients, such as growth factors and other proteins.
Thawing at 37°C in a heated water bath
Frozen serum can also be thawed in a 37°C water bath, however, any time serum is placed in a heated environment it is imperative that the bottles are continuously monitored as they thaw. A shaking water bath is best. If serum is thawed in a heated bath that is not equipped with a shaking apparatus and is not swirled or mixed regularly, the concentrated lipids and proteins can congeal at the bottom of the bottle resulting in a jelly like substance that will not re-suspend. In this case the serum may no longer suitable for use. This is of utmost importance when heat inactivation is employed. Make sure that the water level in the bath is high enough to cover at least 2/3 of the frozen bottles but never comes close to the bottom of the closures. Low water level may cause the serum in the bottom of the bottle to be over heated and become denatured, especially in a bath without shaking capability. If the water level is too high it may get under the bottle cap and contaminate the serum when it is poured.
As the serum begins to thaw, swirl the contents every 10 minutes to prevent protein concentration at the bottom of the bottle. When proteins concentrate at the bottom, denaturation is more likely to occur. Remove the bottles as soon as the serum is thawed, swirl the contents thoroughly and allow cooling to room temperature.
Heat Inactivation
Heat inactivation is a process in which finished serum is placed in a heated water bath and maintained at a temperature of 56°C for 30 minutes. This is the most common method and it was used in the past to inactivate the complement system for immunoassays. Heat activation has also been reported to inactivate other undetermined inhibitors of cell growth in culture, however, the practice is labor-intensive and expensive. The protocol must be followed exactly as too high a temperature or too long a time may destroy some growth factors.
If a customer is adamant about heat inactivation we can perform it, but we do not recommend heat inactivation of fetal bovine serum or any other animal serum that is being used for cell culture. In fact, it can have detrimental effects on the serums ability to support cell growth as well as causing components to precipitate out giving the appearance of contamination.
The great majority of customers who use heat inactivated serum do so because it has been included in protocols that originated years ago and have been carried forward. Heat inactivation was believed to destroy a component in serum called complement. It was also believed to kill adventitious agents such as viruses and mycoplasma, but when collection methods and filtration processes were improved along with the ability to gamma irradiate serum, these issues became irrelevant.
Anyone currently heat inactivating serum should test samples side by side with normal serum and determine if it is really necessary for your particular application. If it is determined that heat inactivation is necessary, then it should be performed using a reliable, repeatable procedure.
Heat Inactivation: Most Accepted Procedure
If the serum was thawed in a refrigerator it should be allowed to come to room temperature prior to placing in a 56°C water bath. If serum is thawed in a 37°C water bath, it may be placed directly in the 56°C bath. It is critical to thoroughly swirl thawed serum until it is uniform in color or denaturation of the proteins will occur during the heat inactivation process. Denatured protein resembles a jelly like substance usually at the bottom of the bottle. If this does occur, the serum can be decanted and may still be used depending on how much denaturation has occurred. Denatured protein should not be confused with Fibrin, a fine precipitate sometimes seen in thawed serum. Fibrin precipitate is a fine whitish powder or flake, which has no ill effects on cell culture.
- Obtain a bottle of similar material and volume and fill with water.
- Suspend a thermometer capable of reading 56°C in the bottle. This will be used as the control.
- Adjust the temperature of the water bath to 56°C and allow the bath to come to temperature.
- Swirl the bottle(s) before placing in the 56°C bath.
- Place the bottle(s) in the 56°C bath along with the control.
- Be sure to swirl every bottle thoroughly every 10 minutes.
- Monitor the temperature in the control and when it reaches 56°C ± 1°C set a timer for 10 minutes.
- After 10 minutes, swirl the bottles thoroughly and reset the timer for 10 minutes.
- Repeat for a total of 30 minutes at 56°C ± 1°C.
- After 30 minutes, remove the bottle(s) from the bath and cool to room temperature.
Charcoal Treated Serum
Charcoal treatment of serum is an effective process for removing hormones and steroids. It has also been used for improving immunoassay systems. Charcoal treated, charcoal filtered or charcoal stripped fetal bovine serum are all terms for the same product. It is made by exposing the serum to a mixture of dextran coated activated charcoal for a period of time. This product is typically very expensive and most labs do not use very much so we offer a procedure for preparing charcoal treated fetal bovine serum. There are several different methods but the actual materials and concentrations are very similar. If the volume of serum you need is 10 – 50 mL, the use of 50mL sterile centrifuge tubes makes the process simpler.
Procedure for Charcoal Treatment of Fetal Bovine Serum
Dextran coated Charcoal: Prepare dextran-coated charcoal by stirring 2.5% (w/v) Norit-A charcoal and dextran T-70 (0.025% w/v) in PBS. Use low endotoxin deionized water if available. Store for 18 hrs at 4°C before use. This is good for up to 1 week when stored at 4°C. Make a volume equal to the volume of serum you need.
Be sure to thoroughly re-suspend the charcoal before using. Take a volume of the dextran-coated charcoal equivalent to that of the serum which is to be stripped. This is where 50mL sterile centrifuge tubes work well. Centrifuge to pellet the charcoal. (500 – 1,000 g for 5 minutes).
Decant the supernatant and replace it with the same volume of fetal bovine serum. If you have low endotoxin de-ionized water, use it to re-suspend the pellet. Centrifuge and decant the supernatant again before adding the fetal bovine serum. This will minimize the introduction of endotoxin in the finished serum if that is a concern.
Vortex the tube to thoroughly mix the charcoal with the serum. Incubate for 12 hours at 4°C.
Pass the stripped serum through a pre-filter and 0.45 micron filter before sterilizing through a 0.2 micron filter. Store at -10° to -30°C.
Certificate of Analysis and Country of Origin are available upon request.
