Dear Research friends,
i have one doubt about FBS (fetal bovine serum) storage condition?.. Once you are all heat inactivated your serum and then aliquote 50 ml each to store at -20C . Why? Once you add in to a DMEM, it will be stored at 4C.. Why?.
MY Q is ..? i can store my FBS at 4C after heat inactivation or Not..? Please explain me...?
HI,
I used to store the FBS after inactivation until I use it at -20ºC. If I need FBS for certain applications, I keep it for short periods at 4ºC in order to avoid thaw-freezing cycles. Once added to your culture media you have to keep it at 4ºC because that are the storing conditions of the culture media.
I hope this helps.
FBS is rich in nutrients, carbohydrates and proteins - all of which can be easily degraded by micro organisms for nutrition and growth. Contamination may be the primary reason FBS is stored at -20 degrees. Heat inactivation kills mycoplasmas and other viable microbes but storing large amounts of FBS at 4 degrees may support the growth of a few types of fungi (owing to the nutrient rich environment of serum).
Secondly, as Miguel correctly points out, frequent freeze thaw cycles may also disrupt important protein components within the serum, that are essential for cell survival. Hence serum is stored in aliquots small enough to be used all at once.
You must have noticed that complete culture medium contains 1-15% FBS only (depending on your cell type), and also contains an antibiotic and an antimycotic agent. The low serum content in complete media lowers the chances of supporting unwanted microbial life and the antibiotic/antimycotic ensures that. However, complete media if refrigerated for long periods eventually gets contaminated.
Also, complete media is used up pretty fast compared to serum. Freezing media is also kept at -20 degrees, mainly because it has a higher percentage of serum.
I hope this helps!!
Best Wishes!
If you are going to use in 1 month, store at +4 degrees. For long-term storage should be keep at -20 degrees.
Hi Senthil,
Serum is an excellent source of nutrients for cells in culture because it contains proteins, lipids, salts, vitamins, minerals, amino acids and other components necessary for growth. Serum also protects against toxic effects associated with pH change, the presence of heavy metal ions, endotoxin, and proteolytic activity. Since serum is a blood product and it contains complement that can lead to complement-mediated cell lysis. To eliminate this risk, serum is heat inactivated (at 56⁰C/30 min) to destroy the complement activity but can also, in some instances, destroy heat labile growth factors, vitamins, amino acids, and hormones present in serum
To effectively preserve the integrity of animal serum, it should be stored frozen at temperatures of -10 to -40 °C. After thawing, use the serum promptly. Liquid serum can be stored refrigerated (2 to 8°C) up to four weeks in small aliquotes. To avoid thaw/freeze cycles or long periods of refrigeration (which results in the degradation of serum nutrients and can induce the formation of insoluble precipitates), it is recommended that the serum be immediately dispensed into small, useful aliquots and refrozen for future use.
To any of the cell culture medium either it is MEM, DMEM or RPMI the serum should be added at the time of usage only.
Good Luck
Thank you very much guys, Shiv Shankar Bingi- If i will store serum in refrigerator freezer up to 2 month .., i should use that serum or not.., Serum filtration is essential means Why?. Can I use unfiltered serum for my A549 cell line?.
HI Senthil,
I usually freeze the serum after inactivation in 50 ml aliquots and use it as I need it. Most of the times it has been frozen for more than 2 months. I never filter the serum after inactivation.
Hope this helps.
Hi Senthil.S,
You can freeze the serum FBS in 50 ml tubes for long time storage. But its better to aliquote into small volume like 20 ml or 25 ml. Thaw it and use and then store at 4C. You can keep in 4C for about a month time..
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