Heat inactivation destroys the activity of the complement system in serum. It is used when complement would interfere with an assay or damage cells, for example in immunological assays, in some cultures of immune cells, and for many antibody-based tests. The standard condition is 56 °C for 30 minutes.
This protocol describes how to heat-inactivate fetal bovine serum (FBS), human serum and other sera correctly in the lab. Whether your application actually needs heat-inactivated serum is discussed in Heat inactivation of serum: necessary or habit?
What heat inactivation does – and doesn’t do
It does:
– destroy complement activity. Several complement components, especially C1, lose their activity within minutes at 56 °C.
It doesn’t:
– sterilise the serum
– remove mycoplasma
– reliably inactivate viruses
These are addressed by sterile filtration, testing and, where needed, gamma irradiation.
It also has a cost: heat-sensitive growth factors and other proteins are partly degraded. Heat-inactivated serum can therefore support growth slightly less well than untreated serum.
Materials
- Frozen serum in its original bottle, e.g. FBS or human serum
- Water bath with thermostat, set to 56 °C, large enough for the bottle
- A reference bottle of the same size and type, filled with the same volume of water
- Calibrated thermometer or temperature probe
- Timer
- Ice bath or refrigerator (2–8 °C) for cooling
- Sterile tubes for aliquots
Step-by-step protocol
1. Thaw the serum
- Thaw the serum slowly at 2–8 °C, e.g. overnight in the refrigerator.
- Alternatively: at room temperature, with the bottle removed as soon as it has just thawed.
- Never thaw at 37 °C or 56 °C. Local overheating causes protein aggregation and precipitate.
- Mix by gently inverting the bottle several times; do not shake. While thawing, proteins and salts concentrate at the bottom of the bottle.
2. Prepare the water bath
- Preheat the water bath to 56 °C and check the temperature with a calibrated thermometer.
- The water level must be above the serum level in the bottle, but below the cap.
- Place the reference bottle with the same volume of water and a thermometer in the bath, next to the serum bottle.
3. Heat
- Put the serum bottle in the water bath.
- Start timing only when the reference bottle reaches 56 °C, not when you put the bottle in. A 500 mL bottle can take 15–30 minutes or more to reach 56 °C.
- Hold at 56 °C for 30 minutes.
- Swirl the bottle gently every 5–10 minutes so the temperature stays even.
- Do not let the temperature rise above 56 °C. Higher temperatures increase protein denaturation and turbidity.
4. Cool
- Immediately after the 30 minutes, take the bottle out of the water bath and cool it quickly in an ice bath or at 2–8 °C.
5. Aliquot and store
- Aliquot the serum aseptically into suitable volumes.
- Label as “HI” (heat-inactivated), with date and lot.
- Store at −20 °C. Avoid repeated freezing and thawing.
Common mistakes
| Mistake | Consequence |
|---|---|
| Timing starts when the bottle goes into the water bath | Serum is at 56 °C for much less than 30 min; complement may remain active |
| Water bath above 56 °C or heating far longer than 30 min | More protein aggregation, precipitate, loss of growth-promoting factors |
| Thawing at 37 °C or 56 °C | Protein aggregation; serum is warmed twice |
| No mixing during heating | Uneven heating within the bottle |
| Heat-inactivating as a “safety” step | False sense of security; viruses and mycoplasma are not reliably inactivated |
| Repeated heat inactivation of the same serum | Additional degradation without any benefit |
Precipitate after heat inactivation
Slight turbidity or fine flakes after heat inactivation are common. They consist mostly of denatured lipoproteins and fibrinogen residues and generally do not affect cell culture performance. If the precipitate disturbs microscopy or flow cytometry, you can centrifuge the serum briefly or filter it. Filtering viscous serum through 0.2 µm can be slow; see 0.1 µm vs 0.2 µm sterile filtration.
Heat inactivation of human serum
Human serum is heat-inactivated in the same way (56 °C, 30 minutes). It is particularly common for:
– immunological assays and T-cell cultures with human serum
– assays in which complement-mediated lysis would distort the result
See Human serum for PBMCs and immunoassays.
Ready-to-use heat-inactivated serum
If you need consistent, documented heat inactivation across many experiments, use a pre-treated product:
– FBS Heat Inactivated – US, Australian or South American origin
– Human Serum Heat Inactivated – mixed donors, EU or US origin
– Heat inactivation of other sera from 50 mL as a serum processing service
FAQ
Why 56 °C for 30 minutes?
At 56 °C, the heat-labile complement components (especially C1) lose their activity within minutes. Thirty minutes is a safe margin. Higher temperatures or longer times damage more proteins without additional benefit.
Do I need to heat-inactivate FBS?
Not by default. Most modern FBS lots are low in complement activity, and many cell lines grow equally well or better in untreated FBS. Heat-inactivate when complement could interfere with your assay or cells. See Heat inactivation: necessary or habit?
Does heat inactivation kill mycoplasma or viruses?
No, not reliably. Mycoplasma are controlled by filtration and testing, viruses by donor/herd selection, testing and, if required, gamma irradiation.
How do I know the serum really reached 56 °C?
Use a reference bottle of the same size and volume with a thermometer, and start the 30 minutes only when it reaches 56 °C.
Is the precipitate after heat inactivation a problem?
Usually not. Slight turbidity is caused by denatured lipoproteins and does not affect most cell cultures.
Can heat-inactivated serum be refrozen?
Yes, after aliquoting. Avoid further freeze-thaw cycles of the same aliquot.
Related: Heat Inactivation: Necessary or Habit? · What is Heat Inactivated FBS? · FBS Heat Inactivated · Human Serum · Serum Processing Services
Prices and availability for all heat-inactivated sera on request – request a quote.
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