Frequently Asked Questions
Do you have questions about a cell culture product? Need to know more about our ordering process? Instantly access answers to frequently asked questions below. Or, browse our technical resources online to find detailed information about our products.
Animal & Human Sera
What is the difference between Fetal Bovine Serum (FBS) and Fetal Calf Serum (FCS)?
There is no difference. These are different names for the same product.
Serum derived from blood of bovine fetuses is referred to as Fetal Bovine Serum (Foetal Bovine Serum) or Fetal Calf Serum (Foetal Calf Serum).
How do the different Fetal Bovine Products manufactured by Atlanta Biologicals compare?
All Atlanta Biologicals brand Fetal Bovine Serum products are collected and processed with the same diligent care and following the same stringent procedures, to guarantee preservation of the growth enhancing properties of the serum and reproducible optimal performance for every lot manufactured.
Furthermore, Atlanta Biologicals requires all serum products to undergo rigorous testing to ensure consistent quality and proven performance even for the most sensitive cell culture systems.
Because not all FBS needs are the same, Atlanta Biologicals has developed a range of products that are tailored to specific cell culture requirements. Additional assays beyond the quality assurance and performance testing conducted on every FBS product, such as a hormone assay, are performed for some Atlanta Biologicals FBS products to address user-specific cell culture requirements. Please consult Atlanta Biologicals’ comparative data chart to determine which FBS product is right for you.
Why is serum heat inactivated?
Generally, the objective of heat inactivation is to destroy complement activity in the serum without affecting the growth-promoting characteristics of the product. Removal of complement activity from the serum is not required for most cell cultures, but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if actually required for optimal cell growth. If heat inactivation is required, the process should be carefully controlled to avoid increased denaturation of serum proteins and formation of crystalline precipitates, potentially resulting in excessive loss of growth performance.
Initially, heat inactivation was also used to inactivate microbial contaminants such as mycoplasma. Heat inactivation for this reason is unnecessary, since Atlanta Biologicals’ FBS is triple 0.1µm filtered and all Atlanta Biologicals brand sera must test negative for mycoplasma, bacteria and fungi.
Sometimes, heat inactivation is performed to disrupt susceptible viruses. In most protocols for this application, prolonged heat inactivation is required. This is not recommended, since valuable components of the serum are rendered ineffective by this treatment.
Should I use heat inactivated serum?
Many laboratories heat inactivate their serum before cell culturing, because it is specified in their protocol. In fact, for many cell culture applications, heat inactivation may be unnecessary. Furthermore, warming the serum-containing medium to 37˚C prior to use, as is the practice in many cell culture laboratories, is often sufficient to inactivate heat-labile complement factors.
Heat inactivation, in general, decreases the growth promoting properties of the serum and causes increased formation of crystalline or flocculent precipitates (salts, proteins, lipids, fibrin…) in the serum.
Since every cell type has different growth requirements, the choice of whether to heat inactivate your serum or not must be made by the researcher. If you have concerns about whether to use heat inactivated serum with your cell line or not, perform a side-by-side comparison of cell growth with heat inactivated versus non-heat inactivated serum.
However, excessive heat inactivation of serum should be avoided whenever possible. If heat inactivation is required, closely monitor the process to avoid overheating the serum or heating over an extended length of time.
When heat inactivation is performed improperly (contents of the bottle are not properly mixed before and during the heat inactivation, serum is heated at temperatures above 56˚C or for more than 30 minutes, usage of non-circulating water bath, water level not up to the level of the serum,…), a much greater amount of precipitates will form and the growth promoting ability of the serum will be further compromised.
For your convenience, Atlanta Biologicals offers all serum products already heat inactivated, saving you valuable time and hassle.
How do I heat inactivate serum?
The objective of heat inactivation is to destroy complement activity in the serum without significantly affecting the growth-promoting characteristics of the product. Removal of complement activity from the serum is not required for most cell cultures, but may be necessary for cultures that are sensitive to the complement activity. Since heat inactivation of the serum may, to some extent, decrease the growth performance properties of the serum, this procedure should only be performed if actually required for optimal cell growth. If heat inactivation is required, the process should be carefully controlled to avoid gelling of serum proteins and excessive loss of growth performance.
Before starting the heat inactivation process, thaw the serum using one of the procedures described in the Technical Note – Heat Inactivation of Serum. Allow the serum to reach room temperature prior to use.
Follow the steps given below and do not leave the serum in the heated water bath for longer than necessary. Monitor the process to avoid excessive inactivation.
- Thaw the serum following the guidelines outlined in “Storing, Thawing and Freezing Serum” and mix the contents of the bottle thoroughly.
- Heat a circulating water bath to 56 oC. Make sure that the bath is large enough to accommodate and immerse the number of bottles being inactivated.
- Place the thawed serum bottles in the water bath so that each bottle is completely immersed up to the level of the serum. Do not immerse the cap.
- Monitor the temperature in the water bath. When the temperature of the water bath returns to 56oC, start timing the process for 30 minutes. Agitate the bottles approximately every 5 minutes during the heat inactivation process to prevent gelling of the serum proteins and to promote more uniform heating of the serum.
- After 30 minutes of heat inactivation at 56oC, remove the serum bottles from the water bath and rapidly cool them in an ice bath. Prolonged treatment of the serum at elevated temperatures will cause deterioration of serum components critical for growth of cells.
After I heat inactivated my serum, some precipitates are visible. Why?
Heat inactivated serum may contain some turbidity, flocculent material or crystalline precipitate. This is a normal occurrence with serum products and in no way indicates that the quality of the product has been compromised.
Commonly, this material is composed of fibrin that has converted from the soluble precursor form, fibrinogen, in serum. Atlanta Biologicals collects and processes all sera rapidly at cold temperatures to yield the highest quality serum with excellent growth properties. This rapid cold processing allows some soluble fibrinogen to remain in the serum after filtration which may convert to fibrin upon thawing.
Precipitates found in serum frequently also contain calcium complexes of inorganic serum components and proteins. Lipid serum components may also cause turbidity of the serum product. Incorrect thawing, frequent thaw-freeze cycles, heat inactivation and extended storage at temperatures above freezing will result in a greater amount of precipitates.
The presence of precipitates in serum does not alter the performance characteristics of the product when used as a growth supplement for cell culture. However, it is not recommended to filter the serum to remove these precipitates. Doing so may result in the loss of some serum nutrients and may clog the filter. Instead, if removal of the flocculence is desired, brief centrifugation of the serum in sterile tubes at 400 g is recommended.
How should serum be stored?
Serum is most stable when stored frozen until needed. The recommended storage temperature for serum is between -5oC and -20oC. Storage of the serum below -20oC may result in breakage of the container due to increased brittleness of the plastic bottle at these temperatures. Repeated freeze-thaw cycles may cause a decrease in serum performance. Repeated freeze-thaw cycles should be avoided as they will cause a decrease in serum performance and an increase in insoluble precipitates. Serum should never be stored in a frost-free freezer, since this type of freezer repeatedly goes through defrost cycles that expose serum to freezing and thawing. Once a serum is thawed, it is recommended to use the serum the same day or to divide it into single-use aliquots and refreeze it.
What procedure do you recommend for freezing serum?
Serum should be frozen as rapidly as possible to avoid prolonged exposure of serum nutrients to higher salt concentrations. Water as the first serum component to freeze will become less dense, resulting in other serum components such as proteins and salts to accumulate at the bottom of the container at a higher concentration. Therefore, slow freezing will result in excessive formation of crystalline precipitates.
How should I thaw serum to ensure that the performance is not compromised?
Frozen serum should be thawed rapidly to avoid prolonged exposure of serum nutrients to higher salt concentrations during the thaw period.
Remove the serum from frozen storage and thaw in a water bath with temperatures ranging from room temperature to 37oC. Periodically agitate the bottle during the thawing process to re-suspend the viscous solutes and to avoid the formation of salt, protein and lipid gradients that can lead to excessive precipitation. Promptly remove the serum from the water bath as soon as the serum is completely thawed. Thoroughly mix the thawed serum before it is added to a culture medium or is heat inactivated as serum components such as proteins and salts tend to concentrate at the bottom of the container during thawing.
Thawing of serum at temperatures above 37oC is not recommended. This process may degrade heat labile nutrients, thus compromising the integrity and performance of the product, and can cause increased precipitate formation.
Once serum is thawed, it should be used promptly or divided into single-use aliquots and refrozen. Alternatively, unused portions of thawed serum may be stored at refrigerated temperatures (2 to 8oC) for a short period of time. However, the length of time that serum can be stored refrigerated with acceptable cell culture results varies with different cell lines.
Avoid thaw-freeze cycles or long periods of refrigeration as this can result in a decrease of stability and performance of the serum.
Is serum light-sensitive?
Serum does contain components that are affected by exposure to light. The effect of light on the serum will depend upon the intensity of light, the wavelength of light and the duration of exposure. Normal handling of serum under room lights should have little effect on the serum. High intensity lights should be avoided and long-term storage under light should be avoided. Certain cell lines may be more sensitive to the effects of light on serum than other cell lines.
My serum contains flocculent material. What could it be?
Serum may show turbidity for a variety of reasons.
Freezing and thawing of serum usually causes denaturation and cryoprecipitation of serum components. The more times serum is subjected to freeze-thaw cycles, the more turbidity is noticeable. This type of precipitation can be minimized by refraining from repeated freezing and thawing. If serum is not used all at once, it is recommended to store it frozen, in smaller, single-use aliquots.
High quality serum is collected and processed rapidly at cold temperatures to yield serum with excellent growth properties. This cold processing leaves some fibrinogen in the serum which may convert to fibrin upon storage, thawing, or heat-inactivation. Fibrin can cause the serum to look slightly turbid or may be visible as flocculence.
Flocculent material does not adversely affect the growth performance characteristics of serum. However, if you wish to remove flocculent material present in your serum, the serum may be transferred to sterile tubes and centrifuged briefly at 400 g. Remove the supernatant and discard the pellet. It is not recommended to attempt to filter serum containing flocculent material, it may clog filters.
Why is there lot-to-lot variability in serum?
Serum is fundamentally important to the proliferation and maintenance of cells cultured in a classical cell culture medium, as the medium usually will not support cell survival without supplementation with animal serum – typically Fetal Bovine Serum – or human serum, at a concentration of 5-20%. Serum is a complex mixture of biological constituents, a majority of which have not yet been fully defined. The composition of these serum components naturally varies somewhat from lot to lot.
The best serum lot for you is the one that works for your cells in your specific application, that is manufactured by a reliable supplier you trust, and that fits your budget. Atlanta Biologicals, a manufacturer of serum with close to 40 years of experience, can help you select the serum lot that is ideal for you. We offer a variety of programs such as lot matching, free samples for lot prequalification, extended lot reserves, and free on-site serum storage, geared towards minimizing the impact of serum variability for our customers while providing high quality product at an exceptional value.
Can I still use my serum if it arrives partially thawed?
All Atlanta Biologicals serum is shipped frozen via overnight service and packaged in dry ice. Without a delay in shipping and at the receiving location, it should arrive frozen. If delays occur, you can still use serum that is partially thawed. Thaw completely, mix gently, aliquot into single-use units if desired, and refreeze.
How is off-the-clot Human Serum different from plasma-derived Human Serum?
Plasma-derived human serum is obtained from pooled human plasma that is collected in the presence of an anticoagulant, and then defibrinated. It is generally more consistent than off-the-clot human serum. Off-the-clot serum is produced from spontaneously coagulated whole blood. Typically, plasma-derived human serum will work well for a large variety of cell culture applications, and is less expensive than off-the-clot human serum.
Media & Balanced Salt Solutions
I am interested in a cell culture medium that is not available as a catalog item. What are your minimum requirements for custom manufactured media?
The most commonly requested customizations involve additions, deletions, substitutions, and concentration changes in the formulations of standard classical cell culture media. Atlanta Biologicals’ custom service also includes the manufacture of customer-designed formulations.
If you are interested in Atlanta Biologicals’ custom manufacturing services, contact us to discuss your specific requirements and to receive a quote. We are able to supply you with cell culture media produced according to your specifications, in quantities as low as 6 liters. We appreciate your interest and look forward to discussing your needs.
How long can I use my cell culture medium once it has been mixed with serum?
Serum should be kept frozen to preserve its growth properties. Once serum has been added to liquid culture media, typically at a concentration of 5-20%, the medium is generally stored refrigerated. Over time, at refrigerated temperatures, the serum will gradually lose its cell growth promoting properties. How long this takes depends upon your particular cell line. Some laboratories need to prepare their culture media fresh on a weekly basis to ensure proper growth of their cell lines. Other labs may have cell lines that are less fastidious and may keep their media for a month or more. It is best to determine for every particular cell line what is required for optimal cell growth.
Can I freeze liquid cell culture media?
Freezing of liquid cell culture media is not recommended due to the fact that freezing typically causes precipitation and formation of insoluble salt complexes.
What is the advantage of adding HEPES to a cell culture medium?
Sodium bicarbonate is the most commonly used buffer in cell culture media due to its nutritional benefits and lack of cytotoxicity. Adding HEPES buffer to a cell culture medium will provide supplemental buffering capacity to the medium at physiological pH.
Why do some cell culture media contain Sodium Pyruvate?
Sodium pyruvate is added to many low glucose and high glucose DMEM formulations. Sodium pyruvate can be used by cells as a readily accessible carbon source for energy production and other critical metabolic pathways, bypassing the need to produce it biosynthetically from glucose or amino acids. Some cell lines require the addition of pyruvate to the culture media since they lack the ability to convert glucose or amino acids into pyruvate.
Supplements & Reagents
Why is HEPES used in cell culture?
HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pKa@37oC=7.3, useful range pH 6.6 – 8.0) is one of several biological buffers that can be used to stabilize the pH of cell culture medium. As cells grow and metabolize the cell culture medium nutrients, they typically produce excess acid – that lowers the pH of the medium. This lowering of pH can be detrimental to the continued growth of cells in culture. HEPES provides more “buffering” capacity than the bicarbonate normally found in cell culture medium – thus slowing the decline in pH as cells metabolize the nutrient medium. For some cell lines this extends the time between “feedings” and promotes healthier cell growth. HEPES can show toxicity with some cell lines, so it is not a universal panacea. Other useful biological buffers include MOPS (pKa@37oC=7.0), TRIS (pKa@37oC=7.8), and PIPES (pKa@37oC=6.7).
What is GlutaminePlus, and how does it compare to L-Glutamine?
L-glutamine is an essential amino acid and plays a major role for the growth and function of cells in culture. Although L-glutamine is stable in crystalline form, it has the tendency to degrade non-enzymatically and irreversibly in solution within a short time period. The rate of this L-glutamine breakdown is dependent on pH, temperature and the presence of various anions.
One of the by-products, ammonia, may act as a toxin or growth inhibitor for the cultured cells.
Because of its chemical instability and significance for cell growth, it is important that the delivery of L-glutamine be optimized to each specific cell culture application.
A recommended way of achieving reliable delivery of L-glutamine to the cells in culture is the use of stable derivatives of L-glutamine in cell culture media.
Atlanta Biologicals brand GlutaminePlus, a derivative of L-glutamine obtained by a chemical reaction of L-alanine with L-glutamine, is available in liquid form or as an ingredient in ready-to-use Atlanta Biologicals brand cell culture media (media with stable L-glutamine).
GlutaminePlus is metabolized within the cells to yield L-glutamine plus the second amino acid. This results in more consistent delivery of L-glutamine to cells in culture and avoids toxic buildup of ammonia in cell cultures. This feature can be especially important for ammonia sensitive cell lines.
Cell Separation Products
What is Ficoll™?
Ficoll™ is a neutral, highly branched, hydrophilic sucrose polymer that dissolves readily in aqueous solution. Ficoll™ is a registered trademark held by GE Healthcare (Amersham Biosciences), formerly owned by Pharmacia. One of the most common applications for Ficoll™ is as a component of density gradient media for the isolation of eukaryotic cells.
We stored our Fico/Lite-LymphoH incorrectly in the freezer. Can we still use it?
Fico/Lite cell separation media should always be stored at refrigerated temperatures. Upon freezing any Fico/Lite product, gradients can form. Slowly thaw the solution and carefully mix it. If no precipitation is visible, the product should still be suitable for use.
Can I dilute my Fico/Lite-LymphoH and use it for separation of human platelets?
No, this is not recommended. Fico/Lite-LymphoH is a ready-to-use solution with a density of approximately 1.077. It is also osmotically balanced for use on specific eukaryotic cells by the addition of an appropriate lower molecular weight chemical. Properly balanced cell separation products will usually have a specific, narrow osmolality range that supports the separation of the desired cell type. Atlanta Biologicals offers Fico/Lite-Platelets, a ready-to-use product for the separation of human platelets.
How can I place my order?
You can place your purchase orders online, email your order to firstname.lastname@example.org, fax in your order to 800-780-7374, or place your order over the phone at 800-780-7788.
For credit card orders, please use our secure online ordering system, or call us at 800-780-7788 to place your order.
What forms of payment can I use?
Atlanta Biologicals accepts purchase orders for approved accounts and credit card orders via MasterCard®, Visa®, American Express®, and Discover®.