The reason for heat treatment (30 min 56°C) is to inactivate complement that might damage the cultured cells when antibodies against these cells are present at the same time.

I take it you look at human PBMC from apparently healthy donors. These cells are then cultured with autologeous serum, i.e. from the same donor. This serum should not contain relevant antibodies against the autologeous cells, i.e. there should be no auto-antibodies in the serum. Therefore the recommendation for your setting is not to heat-inactivate.

Heat inactivation can also damage growth factors, which are present in the serum. By not heat-inactivating you therefore will have also better culture conditions.

Depending on my experiment results, simple inactivation by heating is not sufficient to protect the cells from apoptosis. I wonder whether you observed any evident cell proliferation or differentiation. More information may be necessary to argument or to discuss.

The serum heat inactivation has the main purpose of inactivate complement. Knowing this and depending on the nature of your experiments, you can choose to inactivate the serum or not..

I agree with Giovanna- heat inactivation is usually with the aim of inactivating complement proteins. This is the standard protocol when using serum in assays. The exception would be if you are aiming to look at complement effects within the serum ie. properties of patient serum itself, rather than using the serum as a protein source for your cells.

I agree with Victoria Davenport . Inactivation of serum by heating is for complement inactivation. Define your purpose of your experiment. serum as a source of complement/protein Lalitha kabilan

The reason for heat treatment (30 min 56°C) is to inactivate complement that might damage the cultured cells when antibodies against these cells are present at the same time.

I take it you look at human PBMC from apparently healthy donors. These cells are then cultured with autologeous serum, i.e. from the same donor. This serum should not contain relevant antibodies against the autologeous cells, i.e. there should be no auto-antibodies in the serum. Therefore the recommendation for your setting is not to heat-inactivate.

Heat inactivation can also damage growth factors, which are present in the serum. By not heat-inactivating you therefore will have also better culture conditions.

I agree with Loems ziegler-heitbrock, too.

Thank you all for your helpful responses. Loems, I agree that there shouldn't be any auto-antibodies but hadn't thought about the denaturing of growth factors. However, I am not sure if other components in the culture medium (RPMI+ Glut+Pen-Strep) might cause the formation of membrane attack complexes, thereby lowering my viability. It is not an overly long incubation (

I think there is no evidence to support the idea that these components of the culture medium can induce MAC formation. Suggestion to go ahead and try to culture without heat-inactivation and see what you get in your system.

Heat inactivation step at 56 C for 30 min is to inactivate the compliment systems.

If you are having cytotoxicity, please check the dilution. It is recommendaed to use 2-5% inactivated autologous serum in the experiments.

In PBMCs, you would see lot many cell types. Cells like neutrophils are fasted to dye. However, such neutrophil cell death should also be seen in control conditions (FBS or no-heat activation). My recommendation is always to keeps these controls in your experiments.

Its better to perform experiments in purified lymphocytes, where you will find lesser variability (response and cytotoxicity).

good luck

For reference please see below mentioned article.

J autoimmunity. The role of TH17/IL17 on eosinophilic inflammation.

Supplementary article would be useful for you

Usually I inactivate AB serum obtained by blood donors (30' at 56°C) to culture PBMC and the aim as already said is to inactivate the complement to avoid the damage of the cells in case of antibodies against them eventually present in the serum. So the decision to inactivate or not depends on the culture system you are using and the purpose of your culture. Are you sure that in your autologous system auto-antibodies are not present? Another important issue is the concentration of the serum in the culture, the most frequent is 10% but the range is usually between 2%-20% depending on the aim of your experiments.

As previous responses suggested heating of serum inactivates complements and reduces non-specific cell activation. The common approach is to heat at 56 °C for 30 minutes. But, for certain experimental conditions extended inaction time up to 60 minutes are used. The serum source is also critical as the concentration of lipopolysaccharide (LPS) and other contaminants in the fetal bovine serum may vary from vendor to vendor, and batch to bach. This may lead to non-specific cell activation and high backgrounds. In mixed human PBMC lymphocyte proliferation studies AB human serum or autologous serum should be used to avoid activation by xenogeneic antigens. Heat inactivated serum should be used.

I also did experiments that required me to inactivate serum at 56 to 60 degrees Celsius for 30 min and I observed that all complement-based activities I wanted to be investigated were affected and altered compared to those that were not inactivated. Though I am no really familiar with your experimental protocol, is there really a need to inactivate your serum? Can you set up a related experiment in order for you to establish a baseline or information from which you can refer on your decision whether there is a need to inactivate your serum and from that info you can also determine the right temperature or duration of serum inactivation that supports the growth of your PBMC culture. Good luck.

I really like to know the real purpose of heating the serum, because it is well documented that the first compounds of the complement cascade are very unstable even at -20°C that if you keep it at room temperature then you might loose those first molecules and the fixation will not happen.