What is the best way to thaw serum?
Serum is thawed at 2-8°C to prevent degradation; alternatively, it may be thawed at room temperature with periodic agitation to resuspend the contents.
Thawed serum should be mixed thoroughly before being added to cell culture medium.
Temperatures above 37°C can compromise serum performance by accelerating the degradation of critical serum factors, and should be avoided in the lab.
What are the precipitates in the serum?
Turbidity and flocculent material may be present after thawing or after prolonged freezing and/or refrigeration. Our experience indicates these modifications do not affect the biological performance of the serum.
- Some of the precipitates are due to fibrin:
Our collection procedures and rapid freezing may leave some fibrinogen in the serum. Fibrinogen is the soluble precursor of the clot-forming protein, fibrin. The fibrin may appear after thawing or heat inactivation. Fibrin does not alter the capacity of the serum to promote cell growth. It is recommended to use the serum without treatment (filtration or centrifugation).
- Some of the precipitates are due to Calcium Phosphate:
Serum that is incubated at 37°C for extended periods will become cloudy and deposits may appear. They are composed of calcium and phosphorous. To the best of our knowledge, this does not alter the performance of the serum in cell cultures.
Is it necessary to heat inactivate serum?
For most cell culture applications, heat inactivation of serum is not recommended. Heat inactivation degrades complement proteins that may interfere with immunological assays. Heating serum for prolonged periods of time can reduce or destroy growth factors, as well as increase the formation of deposits which are commonly mistaken for microbial contamination.
How should the serum be heat inactivated?
- Start by thawing the serum and the control serum bottle at 2-8°C.
- Stir the bottled serum to avoid a precipitant from forming during the thawing, then place it in the water bath adjusted to 80°C.
- Once the control bottle temperature reaches 50°C, adjust the bath thermostat to 70°C; when the bottle temperature reaches 55°C, change the thermostat to 60°C.
- When the serum reaches the temperature of 56°C, use a timer (30 minutes is the registered time) and change the thermostat of the water bath to 56°C. During those 30 minutes, check the temperature regularly and maintain it stable by adjusting the thermostat, by adding some cold water, and by lowering the lid or not. Stir the serum every 5 to 10 minutes.
- Keep the serum at room temperature for 30 minutes, and then freeze the serum at -20°C.
. The stirring is crucial for good serum quality. If the serum is not correctly mixed, a precipitant of salts, proteins and lipids will appear, and allow crystalline and flocculation deposits to form. These deposits are not toxic for cell culture but affect the serums appearance and consistency.
. Heating the serum for long periods can destroy the growth promoters, which can also enable deposits to form.
How long can the serum be stored between +2 to +8°C?
Serum may be stored between +2 to +8°C for up to 8 weeks without diminishing its performance (*).
Internal studies in Biowest have shown that during 26 weeks of storage (between +2 and +8°C) the serum's capacity to promote cell growth was not significantly altered.
(*) Art to Science, Vol.19, No.2, "Serum Stability at Refrigerated Temperatures (2-8°C).
What is the recommended storage temperature for the serum?
The serum should be stored frozen and protected from light. The recommended storage temperature is -10°C to -40°C. At temperatures below -40°C, the bottles may become brittle increasing the risk of breakage.
Is it normal that the trypsin products do not always have the same color?
Some preparations of trypsin contain phenol red. Since the product is shipped with dry ice, there could be a significant CO2 build up in packaging. This CO2 may enter the solution and lower the pH slightly, giving an orange (around pH 6.5) vs. pinkish (around 7.3) color. The solution, if orange (acidic) can still be good to use as is, or sodium hydroxide may be added to adjust the pH.
Implication of Tetracycline for FBS in cell biology
Tetracyclines are a family of broadspectrum antibiotics that inhibits bacterial protein synthesis by blocking binding of aminoacyl-tRNA to the ribosome. Due to growing concerns surrounding development of antibiotic resistant microbes, the use of tetracycline as a growth promoter was banned in EU in the early 1970s. However, in the rest of the world tetracycline is still among the most frequently used antibiotics in livestocks. Since 1992, the Tetracycline-controlled Transcriptional Activation system, or “Tet-Off” and “Tet-On” systems as they are often referred to, has been used for induction of mammalian expression in cell-line models used across laboratories worldwide. In the Tet-Off system, gene expression is silenced if tetracycline is present and induction of expression is done by removal of tetracycline from the media. Vice versa, in the Tet-On system, gene expression is silenced in the absence of tetracycline and induced by addition of tetracycline to the cell culture media.
Both systems are very sensitive to even small amount tetracycline found in FBS. As such, if a researcher uses these tools to control gene expression in his cell-culture models, it’s very important have a supply of FBS that is completely free of tetracycline.
Please feel free to contact your biowest representative for further information or guidance in selecting the best suitable batch for your tetracycline sensitive application.