Heat inactivation of FBS is practiced in many cell culture laboratories worldwide. It has become more of a tradition than a scientific principle, many young researchers cannot categorically say why they inactivate serum. They simply do it based on passed over protocol or knowledge.
Culled from gembio.com " At one time, a chief concern was heat-labile immune complement. However, Triglia and Linscott (Journal of Mol. Immunol., vol. 17, pp 741-748, 1980) found that fetal bovine serum in fact contained very low levels of complement. Even in undiluted serum, they found no evidence of hemolysis from complement activity. Moreover, any residual complement was destroyed by thawing fetal bovine serum at 37ºC prior to use (a common step)"
Please share with us why you think this step is necessary or otherwise.
Heat inactivation is for inactivate the complement. It might be absolutely necessary for immune cells culture. It might not be necessary for some others cells.
I do heat inactivation for my cells.
Heat inactivation of FBS is still a controversial issue - some lab do it as routine practice, others argue, that heat inactivation may destroy valuable vitamins, amino acids, growth factors, etc.
Enclosed please find a folder for your information.
Fantastic Gerhard, thank you for sharing the document. I read it some months ago and could not locate it in my drive folder. It is an interesting article indeed!
Unless you are using precious material, why not do a side by side comparison with your cultures?
I always heat inactivate serum, because of the complement issues mentioned above. However, I do not only use FBS, I also use human serum and this definitely needs to be inactivated, therefore to ensure uniformity across experiments, I HI all sera I use. I agree with the comment regarding disruption to proteins, but for my experiments the protein is there, together with the glucose etc. in the buffer as a source of nutrition - all actual experiments are performed in serum free or low serum medium, so unless you undertake your actual data acquisition in high serum, the HI should not cause you a problem, but may save you some - test the question yourself, run the parallel experiment in your cells and let us know the outcome.
Heat inactivation of FBS from companies like GIBBCO ( south american origin) I never did. It works fantastic. However certain other companies FBS we need to do. Generally companies like GIBBCO and Sigma give with heat inactivation and filtered FBS.
Best is dont compromise any where in FBS , I recommend use GIBBCO (South American origin) . Its not much costly too. Aliquot into different 50 ml falcon tubes. take out two among them and rest store in -80 degree. Heat inactivate one falcon . Now filter both of them along with media using. Put two different culture dishes along with cells in them (sometime without cells its difficult to understand its effect in plain media).
Keep for 24-36hrs and result will be in front of you., "which is best"?
Thanks
I have used both heat inactivated and not inactivated FBS. In the past when working with human umbilical smooth muscle cells we as a lab decided it was not necessary to heat inactivate since we weren't concerned with complement. In other labs that I've worked in the FBS has been inactivated due to a passing on of protocols. I personally think it depends on the cells that you're working with and needs to be decided on an individual basis.
We use heat inactivated FBS because of the complement issues. You can also perform parallel experiments and check out if it is vital for cells you use.
Dear Yusuf,
This is a good question. The choice of using of heat- vs. non-heat inactivated FBS is determined by the type of cells you are trying to expand in-vitro.
A study done by Hyclone showed some cell types performed better when grown using heat inactivated FBS, while the use of non-heat inactivated FBS was more favourable for other cell types. The study also demonstrated that the performance of cells was affected by the heat inactivation treatment time and temperature.
Please see the attached document for your consideration.
I hope this will help address you dilemma.
Best Regards
Basil
When I grew malarial parasites, I used human serum and human erythrocytes. I used to heat inactivate serum to prevent lysis of erythrocytes due to complement factors present in the human serum.
For mammalian cells, I have not done heat inactivation and have not observed any cell lysis.
Can someone give their expert opinion on whether it is necessary to heat inactivate serum for growing embryonic stem cells.
Dear Kasirajan Ayyanathan.
About embryonic stem cells I`m not sure, but when I worked on Fibroblast L2L9, i cultured them in both medium containing inactivated serum and activated one. I saw that in the flask I pured inactivated FBS, my cells were dying ans lysed. But in the other one, cells were great.
It has probably already been suggested, but do a side by side comparison in your cells. We always inactivate, because we don't always only use FBS and therefore as we need to HI the other sera, the experiment would not be correctly controlled if the FBS too is not heat inactivated. Good luck.
The German collection of microorganism and cell culture DSMZ uses heat treated FBS for all their cell lines when FBS is needed.Heat inactivation of FBS degrade active phase proteins.
Heat inactivation of FBS degrade complement proteins and other essential factors. Degrading complement proteins is essential for immunological studies as complement proteins may interfere with how the host mount an immune response.
According to Coriell institute, you don’t have to inactivate FBS for most of the cell lines. “Occasionally we do find a differentiated cell line that grows better in heat inactivated serum, but for most lymphoblast and fibroblast lines it does not seem to make a difference whether the serum is heat inactivated or not.”
Reference: http://symposcium.com/2012/07/difference-fbs-heat-inactivated-fbs-fetal-serum/
LET'S BE PERFECTLY CLEAR...
Heat inactivation of CALF or ADULT serum >> *NECESSARY*
Heat inactivation of FETAL serum >> TOTALLY **UNNECESSARY**
... a waste of time, money, and heat labile growth factors.
The reason people use do heat inactivation of FETAL serum is because mistaken protocols propagated to the point where no one recognized the error but do not want to take the risk. For those who have been in biomedical science for decades, we see this in many instances... e.g., early in mol biol protocols salmon sperm DNA ("ssDNA") as a carrier got confused with single strand DNA ("ssDNA") and there are weird protocols that confuse these and aren't rational but still work. HI-FBS is another example.
As some suggested test them side by side. In every instance where I know this happens, THERE IS NO DIFFERENCE and heat inactivation is a waste of time, effort, and money ("time is money"). This is such a minor issue that it does not rise to the level of a technical publication in the open literature. If one discusses this with a KNOWLEDGEABLE technical service person in companies over a certain age, they will tell you they do this NOT because it is a good idea but because enough customers request it even though it is a mistake. If one discusses this with a new tech service person, that newbie won't know from experience, having come up during the age of confused protocols and thus doesn't take a position.
The finding that there are TRACE levels of complement in fetal serum also showed that there were no deleterious effects. The original question reveals the suspicion that there is an unnecessary inherited practice. That suspicion is correct.
"In the case of fetal bovine serum, it turns out that the levels of complement proteins are relatively low [ii]. For applications not involving immunological endpoints, the presence of such low levels of complement have negligible impact on serum performance. It should not be assumed that heat-inactivation of fetal bovine serum is needed or even appropriate. The best way to answer this question is to empirically determine in your own laboratory whether, for a given cell culture application, non-heat-inactivated serum or heat-inactivated serum provides the optimal results....
"It has been shown [iii] that mycoplasma and certain viruses (including such known contaminants of bovine serum as bovine viral diarrhea virus, parainfluenza type 3, infectious bovine rhinotracheitis, reovirus type 3, and adenovirus) are rendered essentially non-infectious at the temperatures/treatment times used for heat-inactivation...
"In summary, heat-inactivation of serum is no longer considered to be a requirement for most cell culture applications. If it must be done, due to the particular research endpoints being followed, it is recommended that heat-inactivated serum offered by serum providers be utilized, rather than purchasing non-heat inactivated serum and then leaving this important treatment step to the inexperienced."
ii. Linscott WD Triglia RP. The bovine complement system. Adv Exp Med Biol 137: 413-430, 1981.
iii. Danner DJ, Smith J, Plavsic M. Inactivation of viruses and mycoplasmas in fetal bovine serum using 56 ͦC heat. BioPharm 12: 50-52, 1999.
Louis & others,
Let me start by saying, 1- I am fairly new to cell culture, and 2- I was trained that heat inactivation of FBS is unnecessary, and an outdated protocol. However, in my work with T-cell acute leukemia cell lines, I have learned that at least 4 of 6 I am currently working with REQUIRE HI-FBS. This is indicated in the Coriell Protocol specifics, and I confirmed in my own side-by-side experiments, which incidentally took a long time to confirm, and required me to re-purchase some of the lines. I am absolutely convinced that certain T_ALL lines just don't do well in non-inactivated FBS. They overgrow, start to change morphology, and die. These same 4 cell lines in Heat Inactivated FBS do fine. Definately follow protocols of suppliers, and do your own side-by-side if not sure to confirm.
Best of luck!
Selene.
I don't see any relevent data here, just opinions or links to opinion articles with single sentences describing heat inactivation usage. Anyone have any actual references that point to data comparing various lines with and without heat inactivation?
Heat inactivation of FBS degrade active phase protein in particular Degrading complement proteins which can effect the outcome of the experiment and immune response in general.
I was trained that heat inactivation of FBS is unnecessary, and an outdated protocol. However, in my work with Acute Leukemia cell lines I have learned that at least 4 of the 6 T-ALL, and 2 of the 4 B-ALL cell lines I work with REQUIRE HI-FBS. This is indicated in the specifics of their respective Coriell Protocols, and I confirmed in my own side-by-side experiments, which took a long time, a lot of reagents, flasks & pipets, and ultimately required me to re-purchase several of the cell lines. I am absolutely convinced that certain immune cell lines just don't do well in non-inactivated FBS. They may do fine for the first few days - week or so, but will soon start growing faster than expected (overgrowing media within a day), grow larger than expected, change morphology, and die!
These same 6 cell lines maintained in Heat Inactivated FBS do just fine over the course of many weeks to month & longer.
I highly recommend following supplier protocols, and if unsure do your own side-by-side tests to confirm.
Best of luck everyone!
Selene.
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