WHAT IS FETAL BOVINE SERUM?
Fetal Bovine Serum (FBS) originates from the blood drawn from a bovine fetus in a closed system of collection at a slaughterhouse. FBS has a high content of embryonic growth promoting factors and is the most widely used serum supplement for in-vitro cultures.
WHERE DOES FETAL BOVINE SERUM ORIGINATE FROM?
Fetal Bovine Serum (FBS) and other bovine serum is a by-product of the meat industry. FBS comes from blood taken at the time of slaughter from a bovine fetus when female cows that are slaughtered for the human consumption of meat are subsequently found to be pregnant.
HOW DO I THAW SERUM?
Ideally, removing the bottle of serum from frozen storage and allowing to thaw overnight at room temperature (or 12 hours) is the best way. Homogenize the serum once thawed without making the serum foam. If you are not able to plan accordingly, you can also thaw the bottle of serum in a shaking/heated water bath set to 37?C until completely thawed.
HOW SHOULD THE SERUM BE HEAT INACTIVATED?
Remove the desired bottle of serum from frozen storage.
Begin by thawing the bottle of serum either at room temperature or use a heated/shaking water bath to thaw at 36?C until competed thawed. Be sure to set the shaking controls to slow in order to avoid possible submersion of bottles in water. If shaking water bath is not available, occasionally, swirl the bottle of serum gently to avoid precipitant from forming during the thawing and Heat Inactivation process.
Once completely thawed, turn on the heated water bath (if not already on) and set to 56?C. Place both the bottle of serum and the control bottle with thermometer in the water bath.
Closely monitor the control bottle’s temperature and once the thermometer reads 56?C, use a 30-minute timer and initiate heat inactivation procedure. During those 30 minutes, check the temperature regularly to ensure a stable temperature reading 56?C (+/-) 3?C.
Once 30 minutes has elapsed, remove bottle of serum and control bottle, set aside at room temperature for 30 minutes – 1 hour (once the bottles have cooled), proceed to freeze the serum at ? -20?C.
Drain water bath and clean accordingly in reference with Good Lab Practices.
The use of a heated/shaking water bath is highly recommended to ensure serum is properly mixed and is crucial to maintain serum quality. If not mixed properly, precipitants of proteins, lipids and salts will form. In the event a heated/shaking water bath is not readily available, be sure to gently swirl / mix the bottles of serum every 5-10 minutes to avoid inconsistencies of product quality. Heating the serum for elongated periods of time can destroy growth promoters and can enable deposits to form.
IS HEAT INACTIVATION NECESSARY?
Heat Inactivation degrades complement proteins that could interfere with specific assays. For most cell culture settings and applications, heat inactivation is not recommended. Effects of Heat Inactivation include a reduction in growth factors, increase of deposits which are commonly mistaken for microbial contamination and reduce performance of the serum.
WHAT DOES USDA-APPROVED MEAN?
USDA-Approved indicates that the serum’s origin (orsource) is from a USDA-Approved country. The serum is produced from blood collected in countries that have been approved by the United States Department of Agriculture (USDA) and deemed fit to export ruminant serum products to the United States of America. Eligible countries that export fetal bovine serum into the United States of America include: Australia, Canada, Chile, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, New Zealand and Panama.
ARE DEPOSITS / PRECIPITATES IN SERUM HARMFUL?
On occasion, turbidity and flocculent deposits may be present after prolonged freezing / refrigeration of serum. These deposits have no affect on the biological performance of the serum and are cosmetic.
Multiple freeze-thaw cycles increase the chance for precipitates being present in the serum. Fibrinogen is the clot-forming protein (Fibrin) and appear as white flakes. Fibrin could present itself after thawing or heat inactivating the serum. Fibrin does not alter the performance of the serum and poses no threat to cultures. Commonly, Fibrin is mistaken for mycoplasmas contaminations in lab settings and is not harmful.
ARE THERE BETTER QUALITY FBS ORIGINS (SOURCES) THAN OTHERS?
To the best of our knowledge, the serum’s origin has no influence on cell growth. Commonly, one lot of fetal bovine serum (FBS) may exceed the control of a specific cell line but not for another. Serum quality is specific for each cell line and can vary. These variations may include (but not limited) to: Geographical location, the time of year the blood was collected and dietary factors etc. Peak Serum strongly encourages our customers to test prior to purchasing to ensure total satisfaction.
ARE THERE CATEGORIES OF SERUM?
Yes, bovine serum is categorized according to the age of the animal from which the blood was collected.
- “Fetal Bovine Serum” originates from fetuses
- “Newborn Calf Serum” originates from calves ? 3 weeks of age
- “Calf Serum” originates from calves between ? 3 weeks – 12 months of age
- “Adult Bovine Serum” originates from cattle ? 12 months of age
DOES GAMMA IRRADIATION AFFECT SERUM PERFORMANCE?
When serum receives a standard dosage of gamma irradiation, no initial loss of performance is observed.
Gamma Irradiation kills viruses, it also effects proteins and reduces the hemoglobin level in the serum which can modify the physical appearance of the serum.
However, gamma irradiation decreases the serum metabolites and could slightly affect grow promotion, cloning and plating efficiency with some cell lines. These possible observed results can vary depending on specific applications.
IS THERE A RECOMMENDED STORAGE TEMPERATURE FOR SERUM?
Storage temperatures of -10?C to -40?C are recommended for serum storage. Temperatures below -40?C may allow the bottles to become delicate and increase the risk for breakage. It is important to avoid direct exposure to light.
WHY IS SERUM GAMMA IRRADIATED?
On occasion, gamma irradiation is needed to eliminate all potential virus risks in serum.
WHAT IS ENDOTOXIN?
Endotoxin is a complexed lipopolysaccharide (LPS), a toxic substance found in the outer cell membrane of most gram-negative bacteria. The bacteria releases endotoxin into the culture environment while growing and upon cell death.
Studies have shown that endotoxin levels may possibly affect the growth or performance of some cultures in specific applications but does not affect all cells equally.
Industry standard for endotoxin in fetal bovine serum (FBS) is ? 10 EU/ml with the highest-grade reporting at ? 1 EU/ml. Peak Serum provides low-endotoxin FBS but generally this is not necessary for many cell lines and applications. We are happy to offer a wide range of cost effective FBS products to help personalize your serum needs.
WHAT IS HEMOGLOBIN?
Hemoglobin is a protein in red blood cells found in finished fetal bovine serum (FBS). Cell fractionation can occur during transportation/handling and processing of raw fetal blood. This can alter the appearance or color of the finished FBS. Generally, normal levels of total hemoglobin in FBS range from 10 – 30 mg/dL and higher concentrations have virtually no effect on cell culture or the quality of the FBS.
WHAT ARE MYCOPLASMAS?
Mycoplasmas are very small bacteria lacking cell walls that belong to various genera within the class Mollicutes. They are the smallest-free living organisms and most commonly discovered in research laboratory settings where cell culture is present.
Peak Serum’s filtration scheme utilizes triple 0.1-micron membrane sterile filters that effectively remove mycoplasma from our serum. Every lot endures stringent testing for mycoplasmas after final filtration and prior to final release to ensure your research is protected. Our serum is tested for mycoplasmas by 9CFR 113.28 for 28 days.
WHY IS MY SERUM CLOUDY?
Serum that is incubated for extended periods of time (heated water bath > 37?C) may become cloudy and deposits may appear. This is caused by calcium and phosphorous and to the best of our knowledge, does not affect or change the performance of the serum in culture.
WHAT IS THE BEST METHOD FOR ADAPTING CELLS TO NEW SERUM LOTS?
The following is a detailed outline on how to best adapt cell lines to new lots of Fetal Bovine Serum. It is the responsibility of the individual to determine the best method per each unique situation. Minor adjustments to stated protocol are always cell dependent for the full protocol.
Cell Culture Adaptation to new Fetal Bovine Serum
- Purpose: To adapt cell lines to new lots of Fetal Bovine Serum
- Responsibility: It is the responsibility of the individual to determine the best method for adapting cells to new serum lots. Minor adjustments to stated protocol are always cell line dependent. Passaging cells according to a strict schedule ensures reproducible behavior and allows evaluation of the health status of cultured cells.
- Definitions: Sub culturing or passaging – the removal of medium and transfer of cells from a previous culture into fresh medium or a procedure that enables further propagation of a cell line.
- Equipment: Equipment of appropriate design for cell culture operations including but not limited to; incubator, laminar flow hood, microscope, pipet aid, etc.
- Materials: Culture vessels containing cells in healthy phase of growth or cryopreserved cells that are at least 90% viable prior to cryopreservation. Consumables and disposables used in cell culture operations including but not limited to; liquid media and additives, serum, sterile disposable culture vessels and liquid handling utensils, gloves, etc.
- Precautions: Wear appropriate personal protective equipment. Use proper aseptic technique.
7.1. Media Preparation
7.1.1. Prepare appropriate culture media with additives depending on cell type.
7.1.2. Supplement cell culture media with a mixture of previously established fetal bovine serum and the new lot of Regeneration Biology Serum FBS.
220.127.116.11. Use a mixture of the old serum and Regeneration Biology Serum FBS.At a minimum, start with a 50/50 blend for several passages and observe the health. If satisfactory, use the new serum for several additional passages. If your cells are easily grown in most lots of FBS then they may not require as much time when compared to cell lines that are known to be more difficult to grow which will require longer to adapt.
18.104.22.168. Finicky or difficult cells should be started in a 25/75 mixture of new too old for several passages. Increase the new serum by increments of 25% for several passages until 100% new serum is achieved. Cells should be gradually introduced to the new serum as opposed to all at once.
22.214.171.124. Maintain the cells in the appropriate mixture of new to old serum until the cells look healthy before increasing the ratio of new to old. This is up to the individual to determine.
126.96.36.199. Once the cells become adapted, it will not be necessary to repeat the adaptation process when switching lots of Regeneration Biology Serum FBS.