What is the point of filtering FBS/FCS before applying the cells to media and after heat inactivation? Is it just to remove small particles/debris or for the removal of microorganisms, as well? Isn't it sterile in its package?
I believe it is simply to remove large particles (flocculence?), such as fibrin. Centrifugation may be a better (and cheaper) option than filtering. See this link:
http://tools.invitrogen.com/content/sfs/brochures/F-13267_Gib_FBS_Faqs_Final.pdf
afterFBS freezing and thawing you have some aggregate and fibrin in you FBS, that in microscopic view are seen but FBS is strile !.therefor FBS fiteration isnt necessary but after medium preparation , FBS and antibiotic addition, it is better to do filtration because it will decrease contamination risk and medium will be so clear.
Best wishes
I am kind agree with the above responses. FBS is sterile from the vendor(s) but contains small particulates when you put it under microscope. We filtered after mixing it with culture medium so that the particles will not affect the images for the in vitro angiogenesis assay. I have tried once not to filter it and the a lot of particles and even small crystals were seen when I tried to image my assay.
me too agree with the previous answer. The procedure I use is the following: Thaw 500ml of FBS @37C, heat inactivate and aliquot in 50 ml tubes. When I need one, I thaw @4C and filter it and then add to the medium (all of this in sterile condition). In this way there's both re-sterilization (just to be sure, in handling you could accidently contaminate) and elimination of small particles/cristal. My cells behave a lot better with filtration :)
We too do the similar things mentioned above. Thaw the bottle and heat inactivate. Aliquot into sterile falcons. Store them back in -20C.
When needed thaw, spin so that the aggregates settle down and use it. We don't additionally filter also most of the times and atleast I have never seen any contamination yet. Its already sterile and the aggregates only are gotten rid of by centrifugation, they don't contaminate!
FBS in the major source of contamination, so it is better to make sure the sterility even though it comes sterile :). and, always aliquots if you have bought it in large volume, only use what is necessary so we can avoid repeated thawing and freezing thus reduce risk of contamination. we always prepare the complete media (FBS+media+glutamax+penstrep), and filter them. make sure the FBS aliquots in the tube is cap tightly & sealed by parafilm :)
Stuart Iain Jenkins;Mehrdad Gholamzad;Ru-Jeng Teng ;Fulvio Celsi; Kalpita Karan ; Juani Mazmin Husin;:
i have one more doubt about FBS.., How many Freeze thaw cycle is recommended?.. Once i was heat inacvtivated my FBS, what happens on that stay..? after that why i put FBS in -20C ? i can keep FBS in 4c means ...? what happens?
In general, it's good practice to use as few freeze-thaw cycles as possible to limit associated degradation of material.
If you can avoid them altogether, then do so. But, the trade-off is a shorter shelf-life.
I doubt anyone can give you an upper limit, the maximum number of cycles 'allowed'. The amount of degradation occurring will vary too greatly between different batches and storage conditions (defrosting/storing at -4C or 25C, exposure to light etc.).
Generally, higher temperatures and stronger (UV) light will result in increased degradation rates, but these vary between molecules.
Also, different cells/assays will be affected differently (or not affected) by the degradation of different constituents of the medium. Loss of growth factors may be critical for some cell types, but not others.
My experience: I defrost 500 ml serum on arrival, aliquot at 50 ml, freeze at -20C, then defrost and use as needed, up to 3 years later. I've also used out of date serum on occasion for pilot experiments, without obvious adverse effects. So, 1 freeze-thaw cycle, even with expired serum, doesn't *obviously* adversely affect culture of rat glial cells. Any extrapolation from this is guesswork, but perhaps other people will offer their experience.
Sigma-Aldrich have a useful technical report:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/SAFC/Bulletin/t010.pdf
This old (1995) paper discusses some elements of serum stability:
http://edoc.hu-berlin.de/oa/degruyter/cclm.1995.33.4.231.pdf?origin=publication_detail
"All measurable quantities examined were stable for four days in separated
serum at 9°C; however, values of inorganic phosphate, uric acid, bilirubin, triacylglycerols, HDL and LDL-cholesterol and creatine kinase were not sufficiently stable when serum was stored at room temperature."
dear Theodore Kapellos,
I ever accepted the FBS from the company, and I found some particle, exactly is Ok. I used it and did not get the contaminate. If you filter it, probably the 'important' molecule in the medium will be lost. the different case, if your FBS already contaminate and filter is not function. just use new FBS.
Don't freeze-thaw several time your FBS, please make some aliquot.
dew
I have always been told that FBS should not be filtered because you may loose some important particles (I imagine that they are talking about some big proteins maybe), what do you think?
Hi
i am not filtered my FBS but my cells culture media became yellow and I looked at my cells but its totally fine do any one have idea about it and explain to me why its so?
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