What are the benefits and disadvantages of using heat-inactivated FBS in cell culture?
- Heating inactivates complement. Active complement can participate in cytolytic events, contract smooth muscle, release histamine from mast cells and platelets, and activate lymphocytic and macrophage cells. Applications where heat-inactivated serum is recommended include immunological studies and culturing of embryonic stem cells (ESCs), insect cells, and smooth muscle cells.
- Heat inactivation helps to achieve bottle-to-bottle and lot-to-lot stability by neutralizing many factors that can vary largely from lot to lot.
- There aren't necessarily disadvantages to heat inactivation of FBS, but there is some evidence that suggests there may be no added benefit to it unless you are carrying out immune studies.
Note: Heat inactivation is performed in a 56 degrees C water bath for 30 min with swirling every 10 min or so for heat distribution and to lower the degree of protein aggregation/flocculant precipitation. Note: If the time or temperature is exceeded, the serum may thicken to a gel. If this occurs, the serum is no longer usable. Unnecessary heat inactivation can take up time and potentially lead to wasted reagents if a mistake is made during the protocol
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1. Pellerin, et al., Bioengineering, published in 2021.
Can you please suggest an alternative to Fetal Bovine Serum, qualified, heat-inactivated, Canada, 500 mL (Cat. No. 12484028) that has been discontinued?
The alternative product is Fetal Bovine Serum, Value, heat inactivated (formerly USDA-approved in North America or qualified, Brazil in other regions), Cat. No. A5256801.
How much of the total protein measured in Fetal Bovine Serum (heat inactivated or otherwise) is attributed to albumin?
Our CoAs only capture the total protein content not specifically the amount attributed to albumin.
How much Fetal Bovine Serum (FBS) do I need to add to my medium?
FBS is added to culture medium at a concentration of 2-10% to provide attachment factors, nutrients, and hormones for mammalian cells, as well as to be a buffer against disruptions like pH changes and endotoxins. FBS has significant amounts of embryonic growth promoting factors like hormones, carrier proteins, and macromolecular proteins. It also has low levels of antibodies and other growth-inhibiting components. For most basal medium, 10% FBS is used. Some applications require up to 20% supplementation, so researchers need to determine what is optimal for their specific application and cell line.
How long can I store Gibco Fetal Bovine Serum at 4 degrees C?
This product can be stored at 4 degrees C for up to 4 weeks. Once the medium has been supplemented with serum, we recommend using it within 2-4 weeks.
How should I thaw Gibco Fetal Bovine Serum?
We recommend thawing the serum overnight at 4 degrees C or in a 37 degrees C water bath, removing as soon as it is thawed. Once thawed, aliquot into single-use sizes and freeze the aliquots. Each aliquot should ideally be thawed only one additional time as repeated freeze-thaw cycles are not recommended.
What temperature should I store Fetal Bovine Serum?
The product is received frozen and should be stored at -5 degrees C to -20 degrees C right away. Do not subject the serum to repeated freeze-thaw cycles.
Can I purchase Gibco Fetal Bovine Serum that has already been heat-inactivated?
Yes, we do offer Fetal Bovine Serum, certified, heat-inactivated (Cat. Nos. 10082139, 10082147, A3840001, A3840002).
How do I heat-inactivate Gibco Fetal Bovine Serum? What does heat-inactivation do?
Heating inactivates complement. Active complement can participate in cytolytic events, contract smooth muscle, release histamine from mast cells and platelets, and activate lymphocytic and macrophage cells. Applications where heat-inactivated serum is recommended include immunological studies and culturing of embryonic stem cells (ESCs), insect cells, and smooth muscle cells.
Heat inactivation is performed in a 56 degrees C water bath for 30 min with swirling every 10 min or so for heat distribution and to lower the degree of protein aggregation/flocculant precipitation.
Note: If the time or temperature is exceeded, the serum may thicken to a gel. If this occurs, the serum is no longer usable.
Is Gibco Fetal Bovine Serum sterile?
Fetal bovine serum is triple filter sterilized using a 0.1 µm filter. All our Gibco cell culture liquid products are prepared by an aseptic process for which each step has been validated to ensure that all products meet the industry standard sterility assurance level of 10^-3; i.e., product that demonstrates a contamination level of no more than 1 of 1,000 units during the manufacturing process. The highest level of sterility assurance (equal to or greater than 10^-6) cannot be achieved without terminal sterilization which is harmful to the performance of cell culture products. Key points of control include validated sterilization cycles for all material with product contact, routine media fills employing bacteriological media, a comprehensive environmental monitoring program, validated cleaning procedures, and a validated final filter integrity testing program. Additionally, filtration and dispensing are performed within positive pressure, HEPA-filtered, environmentally controlled rooms.
Is precipitation in Gibco Fetal Bovine Serum normal?
Since our sera are not pre-aged before filtration, turbidity or flocculent debris may develop during thawing or storage. This flocculence is a normal characteristic of the serum and does not adversely affect performance characteristics of the serum.
Is Gibco Fetal Bovine Serum tested for tetracycline?
No, the tetracycline testing is not being performed for serum lots manufactured after March 1, 2019. This testing can be requested as a custom request.
How can I minimize lot-to-lot variability in serum?
You can test each new lot to be sure that it works for your specific applications. Alternatively, our serum sales team can determine the most consistent, highest-performing serum lot available using the Gibco iMatch Sera Lot Matching Tool.
Can I aliquot and refreeze serum?
Yes, after the overnight thaw, you can aliquot the serum into smaller, single-use aliquots and refreeze it.
Is serum sterile?
Fetal bovine serum is triple-filtered through 0.1 µm filters. Bovine serum is filtered through 0.2 µm filters.
Why should I heat-inactivate fetal bovine serum (FBS)?
Heating serum inactivates complement. Active complement can participate in cytolytic events, contract smooth muscle, release histamine from mast cells and platelets, and activate lymphocytic and macrophage cells. Applications where heat-inactivated serum is recommended include immunological studies and culturing of embryonic stem cells (ESCs), insect cells, and smooth muscle cells.
What does International Serum Industry Association (ISIA) traceability certification for FBS mean?
It means that the company maintains all auditable history of quality and quantity of materials from the point of collection (including specific animal numbers for donor products), and documentation supporting all stages of processing and transportation or commercial transactions.
How long can serum be stored at 2-8 degrees C?
Serum can be kept at 2-8 degrees C for up to 4 weeks.
If my FBS arrives partially thawed, can I still use it?
Our studies have shown that short-term storage of thawed FBS at 4 degrees C for up to 28 days causes no decrease in growth or viability performance. FBS stored at 4 degrees C for longer periods of time should be evaluated on a case-by-case basis should this storage condition be necessary.
Sometimes I notice flocculence in serum. What could it be?
Flocculence may appear in FBS for a variety of reasons. The most common reason is the denaturation of serum lipoproteins. You may observe fibrin, one of the clot-forming proteins present in serum, after the serum has been thawed. This should not affect product performance. To remove the flocculence, transfer the serum to sterile tubes and centrifuge the material briefly at 400 x g. Then filter the resulting supernant along with your media. Do not attempt to filter serum containing flocculence, it may clog filters.
Why does material precipitate in FBS upon storage in the refrigerator?
Gibco FBS is not pre-aged. When stored at 2 to 8 degrees C, the possibility exists for various proteins and lipoproteins in serum (e.g., cold agglutinins, fibrinogen, vitronectin, etc.) to aggregate, and form either perceptible material or observed turbidity. This should not affect serum performance. We recommend that you store FBS at -20 degrees C and avoid repeated freeze-thaw cycles.
How do I know if I need heat-inactivated serum for my cells?
Usually the best way to find out is to go to the source you obtained your cells from. For example, ATCC will have serum requirements for the cells they sell. Most insect cell lines and embryonic stem cell lines require heat-inactivated serum.
Why is the measurement of oxyhemoglobin in serum important/informative?
It is a quality indicator that reflects rapid collection and processing.
How is dialyzed FBS processed?
It is dialyzed by tangential flow filtration against 0.15 M NaCl using a 10,000 molecular weight cut-off membrane until the glucose level is less than 5 mg/dL. Since this is not exhaustive dialysis, low-molecular weight dialyzable components, such as amino acids may not be totally removed. Exhaustive dialysis is not performed because it can result in precipitation and inactivation of serum peptides.
What is BVD virus (BVDV) in fetal bovine serum and how does it apply to cell culture applications?
BVD stands for Bovine Viral Diarrhea. It is one of the most common viral infections in cattle. It is estimated that 70 to 90 percent of the world's cattle population is seropositive for BVD. This virus can cause abnormalities and fetal abortions in cattle. Several strains of BVD exist, some of which are non-pathogenic.
Bovine serum is tested in accordance with 9 CFR, Section 113.53. The BVDV fluorescent antibody test is one of the required tests to meet Title 9 part 113.53 of the Code of Federal Regulations. However, the results of this test are somewhat subjective in the way they are scored. They are scored 0 to +4, based on the level of observed fluorescence. Most samples will have a detectable level of BVDV antigen due to its prevalence in the bovine population. The FA part of the testing can reveal the presence of non-cytopathic BVD strains. The Cytopathogenic and Hemadsorbing Agents testing is used to determine the release of bovine serum, regardless of the BVDV result. We currently report the BVDV results as “Tested”. Any live cytopathic bovine viruses (including the cytopathic BVDV strain) would be revealed in the testing for cytopathogenic agents. In this portion of the testing, the cultures are microscopically monitored for evidence of inclusion bodies, abnormal number of giant cells, or other cytopathology indicative of cell abnormalities. The hemadsorption assay detects the presence of hemagglutinating viruses. The viral hemagglutinin would induce clumping of red blood cells. If a positive result in the cytopathogenic agents or hemadsorbing agents assays is reported, the material would be failed and therefore would not be released by Quality Assurance.
What size packaging is available for your FBS?
Gibco FBS is available in 50 ml, 500 mL, and 1,000 mL bottles and by special order in 3.5 L and 4 L volumes.
At what temperature, and for how long, should serum be heat-inactivated?
You can incubate the thawed product at a thermostatically controlled temperature of 56 degrees C for 30 minutes. Serum can be heat-inactivated in both our plastic and glass bottles. Warning: Do not attempt to heat-inactivate at a higher temperature for prolonged periods as this may compromise the product's performance through protein denaturation.
What does heat-inactivation do to the serum?
The heating process inactivates portions of the complement cascade. Complement occurs in the following events: cytolytic activities, contraction of smooth muscle, release of histamine from mast cells and platelets, enhanced phagocytosis, chemotaxis, and activation of lymphocytic and macrophage cell types.
When should I use gamma irradiated serum?
Gamma irradiation is generally recognized by regulatory agencies as the best method of virus inactivation and for reducing the risk of viruses that are naturally present in animal sera. We offer a small range of gamma-irradiated products ex-stock. Gamma irradiation is also available on request as a custom service for most standard catalog products. We offer a range of fully validated custom processes covering many dose ranges.
What is the importance of gamma-irradiated sera?
Gamma irradiation is recognized as an effective method for inactivating viruses in animal-origin material. Based on USDA regulations for the general requirements for antibody products (9CFR, Section 113.450), the minimum dosage for blood derivatives of animal origin is 25 kGy. Certain European countries require products to be treated prior to importation with a minimum dose of 25 kGy. We will gamma-irradiate serum on request. We have validated a process for utilizing gamma irradiation in the range of 30-45 kGy to inactivate the most common bovine viruses and mycoplasmas that may be present in FBS. The level of inactivation is 6-8 logs for viruses and 6-7 logs for mycoplasmas. We have also demonstrated that physiochemical properties and cell culture performance of serum is not altered by gamma irradiation at levels of 30-45 kGy.
Can I get custom serum products for my specific research requirements?
Yes. We offer a wide range of custom options. Please contact techsupport@thermofisher.com to find your Sera Specialists to discuss your application and research needs. We will work with you to supply sera that is specially processed and tested to meet your individual requirements. Some examples of our custom capabilities are heat inactivation, gamma irradiation, and testing for tetracycline residues.
Do I need to filter the serum before use?
No, there is no need to filter the serum before use. Gibco serum products are manufactured using the most stringent processes, including membrane filtration.
What is the difference between Fetal Bovine Serum and Fetal Calf Serum?
There is no difference in the general use of the terms. Both terms refer to serum from an unborn cow. Fetal Bovine Serum (FBS) was once known as Fetal Calf Serum (FCS).
How much serum should I add to my media?
The serum concentration will vary with the cell line and basal medium used. Please go here to see our recommended sera supplementations for tested cell lines (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/advanced-d-mem-and-mem/recommended-sera-supplementations.html).
Will improper storage of my tissue culture reagents affect the growth rate of my culture?
Yes. Store animal sera at -5 to -20 degrees C. Store media at 2 to 8 degrees C; use within recommended shelf life period. Store complete media (supplemented) at 2 to 8 degrees C, and for complete medium the recommended shelf life is 2 to 4 weeks. Additionally, minimize exposure of sera and media to light.
