Help & FAQs: Cell Culture Products

  • After I heat inactivated my serum, some precipitates are visible. Why?
  • Heat inactivated serum, including Fetal Bovine Serum (FBS) 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. R&D Systems 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. 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 x g is recommended.
  • Can Cultrex® Mouse Laminin I be used on glass cover slips?
  • Coating glass cover slips with Cultrex Laminin I is quite common and has been published extensively.
  • Can I still use my serum if it arrives partially thawed?
  • All R&D Systems Fetal Bovine Serum and other serum products are 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, and the serum becomes partially thawed, the serum can still be used. Thaw serum completely, mix gently, aliquot into single-use units if desired, and refreeze.
  • Can liquid cell culture media be frozen?
  • Freezing of liquid cell culture media is not recommended due to the fact that freezing typically causes precipitation and formation of insoluble salt complexes.
  • How can I harvest my organoids or 3-D cultured cells for subsequent analysis?
  • Organoids or 3-D cultured cells may be harvested using the Cultrex® 3-D Culture Cell Harvesting Kit (Catalog # 3448-020-K)
  • How should cells be cultured prior to setting up the 3-D culture?
  • Cells need to be healthy and actively dividing in 2-D culture. Cells should be passaged two or three times after resuspension from cryopreservation, and they should never surpass 80% confluency during each passage. Cells should also be assessed for viability using trypan blue, and they should exhibit less than 5% staining.
  • How should I determine which Fetal Bovine Serum is most suitable for my cell culture system?
  • R&D Systems 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 Fetal Bovine Serum (FBS) needs are the same, R&D Systems 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 of our FBS products to address user-specific cell culture requirements.
  • 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.Thaw frozen serum at room temperature or in a 37oC water bath. 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.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.
  • 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.
  • How will non-tumorigenic cells/tissues grow or differentiate when implanted in vivo in Cultrex® Basement Membrane Extract (BME)?
  • Non-transformed cells mixed with Cultrex® BME and implanted in vivo have been found to continue to survive and remain differentiated but generally do not grow. No normal tissues have been found to transform under these conditions. For example, Sertoli cells survive at least a week and retain their cord-like structures. 
  • I am interested in a cell culture medium that is not available as a catalog item. Do you offer custom manufactured media?
  • R&D Systems offers custom services, which include the manufacture of customer-designed formulations. The most commonly requested customizations involve additions, deletions, substitutions, and concentration changes in the formulations of standard classical cell culture media. Our custom service also includes the manufacture of customer-designed formulations. We are able to supply you with cell culture media produced according to your specifications, in quantities as low as 6 liters.Visit our custom cell culture media manufacturing website for more details on our services.
  • Is serum light-sensitive?
  • Serum contains components that are affected by exposure to light. However, normal handling of serum under room lights should have little effect on the serum. The effect of light on the serum will depend upon the intensity of light, the wavelength of light and the duration of exposure. Therefore, high intensity lights and long-term storage under light should be avoided. Note that 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?
  • Fetal Bovine Serum (FBS) and other serum may show turbidity and appear to contain flocculent material for a variety of reasons which include the presence of fibrin, and repeated freezing and thawing of serum.R&D Systems serum is collected and processed rapidly at cold temperatures to yield high quality serum. This cold processing can leave trace fibrinogen in the serum which may convert to fibrin upon storage, thawing, or heat-inactivation. This can cause the serum to appear slightly turbid or be visible as flocculence.Freezing and thawing of serum can cause denaturation and cryoprecipitation of serum components. The more 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.Flocculent material does not adversely affect the growth performance characteristics of serum. 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 as it may clog filters.
  • Should I use heat inactivated fetal bovine serum?
  • 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, etc.) in the Fetal Bovine Serum (FBS) as well as in other serum products.  In fact, for many cell culture applications, heat inactivation may be unnecessary.  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 inactivated FBS is frequently used in immunological applications.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.For your convenience, R&D Systems offers all serum with or without heat-inactivation.
  • What are 3-D cultures?
  • 3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or tissue explants are placed within hydrogel matrices, such as Cultrex Basement Membrane Extract, that mimic in vivo cell environments and allow cells to proliferate in three dimensions.
  • What are the differences between Cultrex Rat Collagen I and Cultrex Rat Collagen I, Low Viscosity?
  • Cultrex Rat Collagen I is provided at 5 mg/mL and is not pepsin treated, so it has intact telopeptides and is highly viscous. Cultrex Rat Collagen I, Low Viscosity is provided at 3 mg/mL, and it is easier to pipet. Both are provided in acetic acid and require pH neutralization prior to use. Please consult the provided product data sheets for pH neutralization instructions.
  • What are the different types of 3-D culture?
  • The two principal methods for performing 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex Basement Membrane Extract (BME) or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.
  • What are the variables associated with 3-D culture?
  • The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.
  • What is GlutaminePlus, and how does it compare to L-Glutamine?
  • 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.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.
  • What is Laminin I?
  • Laminin I is a major component of extracellular matrix. Cultrex Laminin I is purified from murine EHS sarcoma. It is composed of α1β1γ1 chains with a total MW of 800,000 Da. Cultrex Laminin I increases cell adhesion, migration, growth, differentiation, neurite outgrowth, protease production, and malignancy. The response is dependent on cell type.
  • What is R&D Systems doing to reduce its use of packaging from non-renewable resources? R&D Systems should consider re-using styrofoam boxes that are in good condition after delivery.
  • Environmental stewardship is important to R&D Systems and its employees. R&D Systems regularly explores "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally and we can identify a recycling center for styrofoam nearest your facility if necessary. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: Doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, Converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and Continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, glass, and styrofoam.
  • What is the advantage of 3-D culture over traditional 2-D culture?
  • While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2-D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo.
  • 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 or Fetal Calf Serum.
  • What is the difference between the standard Cultrex Rat Collagen I and the Cultrex 3-D Rat Collagen I?
  • Cultrex 3-D Rat Collagen I undergoes the same basic purification and efficacy tests as the standard collagen. However, it undergoes additional 3-D culture validation; it has been tested extensively for the ability to promote growth and differentiation of cell types, visualized by morphology in three dimensions in vitro.
  • What is the recommended working concentration for Cultrex Laminin I?
  • The recommended working concentration for thin coating is 0.05-10 µg/cm2 . However, conditions must be optimized for each cell line or model. Cultrex Laminin I should be used at 6 mg/mL for 3-D culture applications.
  • What kinds of tumor cells or biopsy specimens grow in vivo with Cultrex® BME?
  • Many cell lines and tumor biopsy specimens (usually cut into small fragments) have been found to grow in vivo when implanted with Cultrex® BME. These include melanoma, intestinal, prostate, breast, lung, renal, and liver cancers as well as the 3T3 mouse embryonic fibroblast cell line.
  • What procedure do you recommend for freezing serum?
  • Serum, including Fetal Bovine Serum (FBS), 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.
  • What type of analysis is typically applied for organoid or 3-D cell cultures?
  • Within the organoid, spheroid, or 3-D culture, cells may be assessed for morphology, apical/basal polarity, protein localization, and relative proliferation. In addition, cells may be isolated from the 3-D culture and evaluated for levels of RNA and protein expression, as well as modifications to DNA.
  • Which matrix should I use for 3-D culture?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement Membrane Extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.
  • 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.
  • 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 R&D Systems Fetal Bovine Serum is triple 0.1µm filtered and all other R&D Systems manufactured 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.
  • Why is there lot-to-lot variability in serum?
  • Fetal Bovine Serum and other serum products contain is a complex mixture of biological components, a majority of which have not yet been fully defined. The composition of these serum components naturally varies from lot to lot.The best serum lot for you is the one that works for your cells in your specific application. R&D Systems serum manufacturing has a 40 year history of experience, which provides some of the most consistent serum on the market. We can help you select the serum lot that is ideal for your application by offering 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.
  • Which basement membrane protein is best for studying invasion?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes, adipose cells, and migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.

Angiogenesis Assay Kits

  • Are materials supplied in the DIVAA™ kit sterile?
  • Yes, the materials supplied are tested following USP sterility guidelines.
  • Can cells be placed within the angioreactor?
  • While some groups have had success with implanting angioreactors containing viable cells, this application has not been qualified by R&D Systems. Please consult the scientific literature for more information.
  • How do I reduce spontaneous formation of tubular structures on Cultrex BME in the absence of angiogenic factors?
  • Primary endothelial cells, such as Human Umbilical Vein Endothelial Cells (HUVECs) form capillary-like structures in the absence of added angiogenic factors less often than immortalized endothelial cells. Generally, reducing the number of cells per cm2 plated onto Cultrex BME will result in less background or spontaneous tube formation. Titrate the number of cells and find optimal conditions for your specific cell line. When endothelial cells fully form capillary structures in response to angiogenic activators, but not in their absence, you may proceed.
  • What are the variables associated with the Tube Formation Assay?
  • The major variables associated with tube formation are composition of the Cultrex Basement Membrane Extract (BME) hydrogel, thickness of the hydrogel, cell density, composition of angiogenic factors in the assay medium, and assay period.
  • What cell types can be used in the Tube Formation Assay?
  • The Tube Formation Assay is specific for endothelial cells, either primary cells or immortalized cell lines. Only endothelial cells form capillary-like structures with a lumen inside. Other endothelial cell types form other structures.
  • What is DIVAA™?
  • The Directed In Vivo Angiogenesis Assay (DIVAA™) is the first in vivo system for the study of angiogenesis that provides quantitative and reproducible results. Angioreactors (silicone cylinders closed at one end) containing 20 µL of basement membrane with/without angiogenic-modulating factors are implanted subcutaneously in the dorsal flank of nude mice. With the onset of angiogenesis, vascular endothelial cells proceed to grow into the basement membrane extract and form vessels in the angioreactor. As early as nine days post-implantation, there are enough cells to determine an effective dose response to angiogenic modulating factors.
  • What is the difference between the FITC-Lectin and FITC-Dextran in the DIVAA protocol?
  • FITC-Lectin specifically binds to endothelial cells, so it counts the total number of endothelial cells contained within the angioreactor. FITC-Dextran does not bind endothelial cells. Instead, it is dispersed within the blood of the mouse, being evenly distributed within the blood vessels, so it quantitates the total volume of blood contained within the vessels within the angioreactor. Both procedures have demonstrated equivalent results empirically.
  • What strains of mice has DIVAA™ been used with?
  • DIVAA™ has been used with nude mice, the recommended and qualified strain for the assay. The mouse strain C57Bl has reportedly been used successfully with the kit. However, optimization is required, and variability is commonly reported. If you require C57BI mice for your experiments, it is recommended to allow the implantation to go for up to 15 days.
  • Where are the DIVAA™ angioreactors implanted?
  • The angioreactors should be implanted on the host mouse dorsal lateral flanks. These areas exhibit sufficient vasculature to support initiation of angiogenesis, and they are also strategically positioned to prevent removal by the host mouse. Other areas may be used for implantation; however, this is outside of the product specification. Please consult the scientific literature for more information.
  • Which Cultrex Basement Membrane Extract (BME) should I use for the Tube Formation Assay?
  • Cultrex Reduced Growth Factor BME (RGF BME) is generally used for testing compounds that promote angiogenesis because formation of capillary-like structures (tubes) is significantly less compared to non-growth factor reduced varieties of Cultrex BME. The Cultrex In Vitro Angiogeneis Assay (Tube Formation) includes a qualified production lot of Cultrex RGF BME that exhibits reduced background tube formation in the absence of angiogenic factors.
  • Why does DIVAA™ contain a FGF-2/VEGF mixture to promote angiogenesis?
  • While FGF-2 and VEGF have both been demonstrated to promote angiogenesis in DIVAA™, the FGF-2/VEGF mixture provides a synergistic effect allowing a drastic increase in response using less total growth factor.

Basement Membrane Extracts

  • How does Cultrex® Basement Membrane Extract (BME) promote cell differentiation?
  • All epithelial and endothelial cells are in contact with a basement membrane matrix on at least one of their surfaces. By providing them with their natural matrix in vitro as a substrate for the cells that provides biological cues, the cells can assume a more physiological morphology (i.e. correct shape) and begin expression of cell-lineage specific proteins. Two-dimensional plastic surfaces, in combination with serum-containing media, cause cells to flatten, proliferate and de-differentiate.
  • How should Cultrex Basement Membrane Extract (BME) be stored and handled?
  • BME should be stored at or below -20°C for optimal stability. Preparation of working aliquots is recommended. BME should be thawed overnight on ice at 4°C, however long term storage at 4°C is not recommended. Freeze/thaw cycles and gel-liquid phase transitions should be avoided, since they can compromise product integrity.
  • How will non-tumorigenic cells/tissues grow or differentiate when implanted in vivo in Cultrex® Basement Membrane Extract (BME)?
  • Non-transformed cells mixed with Cultrex® BME and implanted in vivo have been found to continue to survive and remain differentiated but generally do not grow. No normal tissues have been found to transform under these conditions. For example, Sertoli cells survive at least a week and retain their cord-like structures. 
  • What are 3-D cultures?
  • 3-D cultures are in vitro cultures where immortalized cell lines, primary cell lines, stem cells, or tissue explants are placed within hydrogel matrices, such as Cultrex Basement Membrane Extract, that mimic in vivo cell environments and allow cells to proliferate in three dimensions.
  • What are the different types of 3-D culture?
  • The two principal methods for performing 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex Basement Membrane Extract (BME) or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.
  • What are the variables associated with 3-D culture?
  • The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.
  • What is the advantage of 3-D culture over traditional 2-D culture?
  • While 2-D culture has been used for studying many aspects of cell function and behavior, the tissue-culture treated plastic environment is unlike anything found within living organisms. As a result, cells in 2-D culture exhibit altered morphology, function, proliferation, and gene expression when compared to their emanating tissues. By placing these cells in a 3-D environment, they assume biological and biochemical characteristics similar to what is observed in vivo.
  • What is the difference between Cultrex® Basement Membrane Extract (BME) and Cultrex® 3D Culture Matrix?
  • Cultrex® 3D Culture Matrix was developed to provide the most standardized basement membrane extract for use in 3D Cultures. A special process is employed to reduce growth factors and provide material at a standard concentration of approximately 14 mg/mL. This material is then incorporated in a 3D culture to validate efficacy. 3D Culture Basement Membrane Extract promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3) into acinar structures. The Cultrex® 3D Culture Matrix is essentially the same as our standard Cultrex® BME, but has been additionally qualified with the functional 3D assay as described above.
  • What is the Tube Formation Assay?
  • The Tube Formation Assay is based on the ability of endothelial cells to form three-dimensional capillary-like tubular structures when cultured on a hydrogel of reconstituted basement membrane, such as Cultrex Basement Membrane Extract (BME).
  • What kinds of tumor cells or biopsy specimens grow in vivo with Cultrex® BME?
  • Many cell lines and tumor biopsy specimens (usually cut into small fragments) have been found to grow in vivo when implanted with Cultrex® BME. These include melanoma, intestinal, prostate, breast, lung, renal, and liver cancers as well as the 3T3 mouse embryonic fibroblast cell line.
  • Which matrix should I use for 3-D culture?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement Membrane Extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.
  • Which basement membrane protein is best for studying invasion?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes, adipose cells, and migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.

Cell Culture Media and Supplements

  • After I heat inactivated my serum, some precipitates are visible. Why?
  • Heat inactivated serum, including Fetal Bovine Serum (FBS) 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. R&D Systems 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. 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 x g is recommended.
  • Can Fico/Lite-LymphoH be diluted and used 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.
  • Can liquid cell culture media be frozen?
  • Freezing of liquid cell culture media is not recommended due to the fact that freezing typically causes precipitation and formation of insoluble salt complexes.
  • Can Mesenchymal Stem Cells (MSCs) grown in StemXVivo® MSC Expansion Media (Catalog # CCM004) be cryopreserved?
  • MSCs grown in StemXVivo® MSC Expansion Media can be cryopreserved using appropriate cryopreservation media, such as StemXVivo® Serum-Free MSC Freezing Media (Catalog # CCM016) or equivalent.
  • 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. 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 it is generally more consistent and less expensive than off-the-clot human serum.
  • How long can I use my cell culture medium once it has been mixed with serum?
  • The length of time a cell culture medium that is mixed with serum can be used depends upon your cell line. This is because medium is generally stored refrigerated once serum has been added to liquid culture media. At refrigerated temperatures, the serum will gradually lose its cell growth promoting properties over time. These properties are best preserved when serum, without cell media, is stored frozen. While some cell lines may require that culture media is prepared fresh on a weekly basis, less fastidious cell lines may tolerate cell culture media stored refrigerated for up to a month. It is best to determine what is required for optimal cell growth for your cell line.
  • How should Cultrex Basement Membrane Extract (BME) be stored and handled?
  • BME should be stored at or below -20°C for optimal stability. Preparation of working aliquots is recommended. BME should be thawed overnight on ice at 4°C, however long term storage at 4°C is not recommended. Freeze/thaw cycles and gel-liquid phase transitions should be avoided, since they can compromise product integrity.
  • How should I determine which Fetal Bovine Serum is most suitable for my cell culture system?
  • R&D Systems 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 Fetal Bovine Serum (FBS) needs are the same, R&D Systems 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 of our FBS products to address user-specific cell culture requirements.
  • 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.
  • I am interested in a cell culture medium that is not available as a catalog item. Do you offer custom manufactured media?
  • R&D Systems offers custom services, which include the manufacture of customer-designed formulations. The most commonly requested customizations involve additions, deletions, substitutions, and concentration changes in the formulations of standard classical cell culture media. Our custom service also includes the manufacture of customer-designed formulations. We are able to supply you with cell culture media produced according to your specifications, in quantities as low as 6 liters.Visit our custom cell culture media manufacturing website for more details on our services.
  • Is serum light-sensitive?
  • Serum contains components that are affected by exposure to light. However, normal handling of serum under room lights should have little effect on the serum. The effect of light on the serum will depend upon the intensity of light, the wavelength of light and the duration of exposure. Therefore, high intensity lights and long-term storage under light should be avoided. Note that certain cell lines may be more sensitive to the effects of light on serum than other cell lines.
  • Should I use heat inactivated fetal bovine serum?
  • 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, etc.) in the Fetal Bovine Serum (FBS) as well as in other serum products.  In fact, for many cell culture applications, heat inactivation may be unnecessary.  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 inactivated FBS is frequently used in immunological applications.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.For your convenience, R&D Systems offers all serum with or without heat-inactivation.
  • 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.
  • What are the different types of 3-D culture?
  • The two principal methods for performing 3-D culture are the top assay and embedded assay. For the top assay, cells are seeded on a thick gel of Cultrex Basement Membrane Extract (BME) or Extracellular Matrix Protein. A thin overlay of cell culture medium is then applied to the cells. For the embedded assay, cells are resuspended within a thick gel of Cultrex BME or ECM and the culture media is applied on top. The top assay is easier to setup, to control seeding densities, and to keep cells within one focal plane for analysis.
  • What are the variables associated with 3-D culture?
  • The major variables associated with 3-D culture are cell type, cell seeding density, composition of hydrogel, thickness of hydrogel, stiffness of hydrogel, composition of cell culture medium, and time of culture.
  • 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.
  • What is GlutaminePlus, and how does it compare to L-Glutamine?
  • 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.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.
  • 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 or Fetal Calf Serum.
  • What procedure do you recommend for freezing serum?
  • Serum, including Fetal Bovine Serum (FBS), 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.
  • Which matrix should I use for 3-D culture?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement Membrane Extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and is commonly inhabited by stationary cells, such as fibrocytes and adipose cells, as well as migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.
  • 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.
  • 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).
  • 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 R&D Systems Fetal Bovine Serum is triple 0.1µm filtered and all other R&D Systems manufactured 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.
  • Why is there lot-to-lot variability in serum?
  • Fetal Bovine Serum and other serum products contain is a complex mixture of biological components, a majority of which have not yet been fully defined. The composition of these serum components naturally varies from lot to lot.The best serum lot for you is the one that works for your cells in your specific application. R&D Systems serum manufacturing has a 40 year history of experience, which provides some of the most consistent serum on the market. We can help you select the serum lot that is ideal for your application by offering 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.

Cell Invasion/Migration Assays

  • Can the Calcein-labeled invasive cells be subcultured?
  • Calcein AM cytotoxicity should be determined empirically for each cell line or model. For best results, the cells should be removed from the cell dissociation solution and placed in fresh culture medium as soon as possible.
  • How do the Cultrex Cell Invasion Assays compare to wound healing assays?
  • Wound healing assays, also known as scratch assays, monitor cell migration laterally on a tissue culture treated plate. This is accomplished by generating a void in a cell monolayer by either removing cells or treating the surface of the plate to prevent cell growth in a designated area. The assay measures the ability of the cell monolayer to fill this void, and it may be conducted in the presence of extracellular matrix proteins. Since this assay is conducted within one chamber, there is no chemotactic gradient, and without the membrane, the cells are no longer required to change shape and squeeze through the pores. Another potential problem is that this assay does not control for differences in cell proliferation. While wound healing assays may be valuable for supplementing the Boyden chamber assay, it is not a replacement.
  • How does Cultrex® Basement Membrane Extract (BME) promote cell differentiation?
  • All epithelial and endothelial cells are in contact with a basement membrane matrix on at least one of their surfaces. By providing them with their natural matrix in vitro as a substrate for the cells that provides biological cues, the cells can assume a more physiological morphology (i.e. correct shape) and begin expression of cell-lineage specific proteins. Two-dimensional plastic surfaces, in combination with serum-containing media, cause cells to flatten, proliferate and de-differentiate.
  • How will non-tumorigenic cells/tissues grow or differentiate when implanted in vivo in Cultrex® Basement Membrane Extract (BME)?
  • Non-transformed cells mixed with Cultrex® BME and implanted in vivo have been found to continue to survive and remain differentiated but generally do not grow. No normal tissues have been found to transform under these conditions. For example, Sertoli cells survive at least a week and retain their cord-like structures. 
  • What is cell invasion?
  • Cell invasion is cell migration through a physiological barrier in response to a chemoattractant, and this recapitulates cell movement within a physiological environment which is composed of extracellular matrix proteins. Cultrex® Cell Invasion Assays evaluate cell invasion based on the cells ability to traverse membranes that are coated with a layer of extracellular matrix proteins. The cells must traverse this barrier through a combination of protein degradation and cellular locomotion.
  • What is cell migration?
  • Cell migration is the movement of cells in response to a chemical stimulus; also known as chemotaxis. Cultrex® Cell Migration Assays evaluate cell migration based on the cells ability to traverse an uncoated membrane with 8 µm pores, in response to a chemotactic gradient. The cells must undergo cytoskeletal remodeling to fit into the pores and pull themselves through to the underside of the membrane.
  • What is the difference between Cultrex® Basement Membrane Extract (BME) and Cultrex® 3D Culture Matrix?
  • Cultrex® 3D Culture Matrix was developed to provide the most standardized basement membrane extract for use in 3D Cultures. A special process is employed to reduce growth factors and provide material at a standard concentration of approximately 14 mg/mL. This material is then incorporated in a 3D culture to validate efficacy. 3D Culture Basement Membrane Extract promotes differentiation of a human epithelial cell line derived from mammary gland (MCF-10A) and human prostate (PC-3) into acinar structures. The Cultrex® 3D Culture Matrix is essentially the same as our standard Cultrex® BME, but has been additionally qualified with the functional 3D assay as described above.
  • What kinds of tumor cells or biopsy specimens grow in vivo with Cultrex® BME?
  • Many cell lines and tumor biopsy specimens (usually cut into small fragments) have been found to grow in vivo when implanted with Cultrex® BME. These include melanoma, intestinal, prostate, breast, lung, renal, and liver cancers as well as the 3T3 mouse embryonic fibroblast cell line.
  • Which basement membrane protein is best for studying invasion?
  • Choice of matrix should correspond to the environment that you wish to recapitulate. Cultrex Basement membrane extract (BME) will recapitulate the basal lamina, which underlie most cells of epithelial or endothelial origin. Cultrex Collagen I is the major constituent of connective tissue, and it is commonly inhabited by stationary cells, such as fibrocytes, adipose cells, and migrating cells, such as mast cells, macrophages, monocytes, lymphocytes, plasma cells, and eosinophils.