Protocol – Heat Inactivation of Serum


  • Heat inactivation of serum refers to the process which involves treatment of serum at a higher temperature to inactivate unwanted active agents in serum particularly serum complement.
  • The recommended temperature for heat inactivation ranges from 45°C to 62°C and time ranges from 60 min to 15 min with the most common treatment condition being 56° C for 30 min.
  • In addition to inhibiting the complement system, heat inactivation is also reported to reduce the risk of microbial contaminants like mycoplasma. However, heat inactivation often results in precipitation, and serum appears translucent. Precipitates in serum are frequently mistaken for microbial contaminants.
  • In addition, inactivation of useful serum components (growth factors, vitamins, etc.) has also been observed. A compromise in the growth properties of some cell lines has also been reported.
  • Serum from embryonic origin often contains low levels of heat-labile complement components. Therefore, heat inactivation of fetal bovine serum or serum from the embryonic origin is not as common as heat inactivation of serum from the adult origin. However, certain applications such as immunoassay may require heat-inactivated serum for best results.
  • Serum from adult animal origin often contains undesirable activity (complement system), which may inhibit or destroy cells under certain conditions.


Equipment and disposables:
♦ Water bath
♦ Centrifuge
♦ Ice bucket with ice
♦ Conical centrifuge tubes (50 ml polypropylene tube)
♦ Laminar flow hood (Certified Class II)

Reagents and solutions
♦ Serum

Starting material:
A 50 ml aliquot of serum in 50 ml polypropylene centrifuge tube

Note: Large sizes (e.g. 500 ml) of serum can also be heat-inactivated. A 50 ml aliquot in a 50 ml polypropylene centrifuge tube is convenient as the precipitated material can be easily removed and can also be used for the preparation of a 500 ml culture medium.

Prior to start:
Set the water bath at 56°C


Precaution: All the transfer of serum from one tube to another one must be done inside the laminar flow hood under aseptic conditions. 

Step 1: Take a 50 ml aliquot of serum from the freezer. Thaw it.
After the serum is thawed, you must invert the tube 2 -3 times to mix all the content of the serum.

1. Thawing is usually done by keeping serum in a 37°C water bath. You can also keep the frozen serum overnight at 4°C for thawing.
2. If the serum is not aliquoted or you can thaw the stock serum bottle and make 50 aliquots in sterile 50 ml polypropylene centrifuge tubes (see protocol).

Step 2: Incubate serum at 56°C for 30 min. Swirl the tube occasionally to heat the tube evenly.

Make sure that the temperature of the water-bath is 56°C.

Step 3: After incubation over, cool the serum immediately by keeping on ice. Store serum overnight at 4°C.

Step 4: Centrifuge at high speed (4000 – 5000 rpm) to remove precipitate.

Store the heat-inactivated serum at -20 °C.

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  1. Begin timing for 30 minutes. Swirl the serum every 5 to 10 minutes to ensure uniform heating and to prevent protein coagulation at the bottom of the bottle. Notes: Heating the serum for longer than 30 minutes or higher than 56°C will have an adverse effect on the efficacy of the serum and will most likely cause an increase in the amount of cryo-precipitate that might form. For volumes less than 500 ml of serum to be heat inactivated, reduce the time of incubation in the 56°C water bath.

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