Heat Inactivation

Protocol for Serum Products​

Heat Inactivation of Serum Products:

The objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from serum, such as fetal bovine serum, is not required for most cell cultures but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if required for optimal cell growth. Researchers should evaluate the applicability of heat inactivation regarding their own application.


  1. Thaw the desired Serum until at room temperature, if thawed in refrigerator allow serum to set at room temperature.
  2. Heat agitating water bath (set at 56°C) and fill with water to ensure bottles will be immersed to cover the level of the serum.
  3. Gently mix the contents of the bottle/serum thoroughly before placing in the water bath.
  4. It is recommended to have a weight to secure each bottle and prevent tipping while water bath is agitating.

Heat Inactivation:

  1. Once water bath temperature reaches 56± 2°C, place the serum bottles in the water bath (include control bottle with thermometer to monitor internal temperature of serum).
  2. Set the water bath to agitate or swirl the bottles every 10 minutes to ensure serum remains uniform. NOTE: Agitation prevents gelling and will promote a uniform heating.
  3. When the control bottle reaches an internal temperature of 56± 2°C, begin timer for 30 minutes.
  4. After 30 minutes, remove the bottles and transfer into cooling rack/space.  Cool the bottles for at least 1 hour before aliquoting or return to freezer.


The process should be carefully controlled to avoid increased formation of crystalline and flocculent precipitates, gelling of serum proteins, and excessive loss of growth performance.  Significant damage to serum can occur when bottles are not agitated/swirled, exposed to higher than required temperatures or heated over extended lengths of time.


Precipitates & Flocculent Material in Serum Products Serum that has been frozen and thawed, or heat inactivated may contain some turbidity, flocculent material, or crystalline precipitates.

This is a normal occurrence with serum products and not an indication that the quality of the product has been compromised.

The presence of these substances in serum does not alter the performance of the serum when used for cell culture. It is not recommended to filter the serum to remove these precipitates. Doing so may result in the loss of some serum nutrients.

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