For the last few months we have been seeing increased background levels of DNA damage signaling by Western blot (spontaneously high FANCD2 ubiquitination) and immunofluorescence (H2AX). We have finally traced the cause to switching to a new batch of FBS. Has anyone ever seen this problem before? I have heard that serum can contain nucleases, but this batch is particularly potent at activating the damage respone - equivalent to 40ng/ml mitomycin c! Is it possible that the batch has a contaminant? Should I be screening batches before purchasing or is this a relatively rare phenomenon?
In a related question, does anyone wish to purchase a 20L batch of FBS, going cheap!? (-:
Hello Andrew,
FBS can induced DNA-Damage as well as cell death. The spontanouse cell death of cell lines in culture variates between 2-30 % using different FBS. The percentage of endotoxine is important. You should use FBS with low endotoxine lower than 1%. In addition, FBS can also block apoptosis induction of anticancer drugs. Bevore we buy FBS, we test al lot of batches. We test different FBS in culture using different cell lines. We measure spontanouse death as well as induction of cell death by cytotoxic drugs. Then we make a reservation of FBS with the same batch, to get comparable results for a long period of time.
Good luck.
You should report this to the manufacturing company... This may produce erroneous results... We also faced similar problem where we observed variations even in the control. Finally we found that the FBS was having problem. We changed FBS source and moved on... ")
I agree with Rangrez about the manufacturing problems and that should be communicated to the companies in order to stop supplying the same batch products to others.
And these are very common issues now a days about the products with more/less activity due to problems in batch operations. We should be very careful about these types of issues in assessing our results.
Variation in background levels is well known from mutagenicity testing/biomonitoring and may sometimes be due to FBS. It is usually much smaller and accounted for by processing cases and controls in parallel. If you have some of your old FBS batch or medium left run your experiment with that in parallel to the new batch, document the difference and report it to the manufacturer. With these data you may ask for a replacement of the new batch and the manufacturer might provide it. Good luck !
Do you mind sharing with us which company supplied your FBS? It is true that FBS, depending on where it is coming from, can have a certain number of different contaminants, but I had never heard of this bad before (we normally measure AP sites). The company should replace your batch for free!
I never heard about this before.
As it is well known that DNA damage occurs by the change in the microenvironment, it is very possible that the FBS you were using contains some DNA damaging agents.
On the lighter side; if you can determine what is there in the FBS causing DNA damage, you can do a simple experiment by using that FBS as a mild DNA damaging reagent with same pH and many other properties.
Please do share the name of the company with the researcher community.
Hello Andrew.
I agree with the rest of the people advice.
I have not observed such an effect previously but it is not surprised me.
We had a big problem with contaminated FBS and since we are a microbiology lab, I am not going to tell you what trouble caused.
It is good to keep monitoring any new batch that you get and inform the company and of course ask for a replacement. Try to buy FBS in numbers so it would come from the same batch.
Good luck with it.
Not directly, but there are some good papers to show how different serums interact with substances differently. e.g. with VAM http://onlinelibrary.wiley.com/doi/10.1002/em.21809/abstract
Hello Andrew,
FBS can induced DNA-Damage as well as cell death. The spontanouse cell death of cell lines in culture variates between 2-30 % using different FBS. The percentage of endotoxine is important. You should use FBS with low endotoxine lower than 1%. In addition, FBS can also block apoptosis induction of anticancer drugs. Bevore we buy FBS, we test al lot of batches. We test different FBS in culture using different cell lines. We measure spontanouse death as well as induction of cell death by cytotoxic drugs. Then we make a reservation of FBS with the same batch, to get comparable results for a long period of time.
Good luck.
Yes you should definitively be screening batches before buying. Especially if testing drugs and toxics (as per Claudia's post).
Most companies (I get mine from LifeTech/Gibco) can provide free 50ml aliquots...
Then when you like it, you buy as much as possible! Good luck!
As a tangential but possibly related comment: With regard to bacterial growth media, I have observed a mutagenic effect of growth in Trypticase Soy Broth that I do not find with cells (Serratia) grown in Nutrient Broth. Seems reproducible. Ingredient/contaminent unknown. Effect on results could be profound.
Thanks everyone for your suggestions. Ironically, we did screen a whole series of FBS batches before buying - but we tested them in more rigorous assays, eg support of primary bone cultures and normal HeLa cell proliferation assays. We never thought to screen for activation of DNA damage signaling as it has never been an issue for us before (over my 10 years of DNA damage research I've never come across this problem!). Reading the spec sheet from the supplier (Bovogen, an Australian supplier - but this serum batch is of French origin), the endotoxin is measured at 0.5EU/ml which is well below the threshold of 30EU/ml for "low endotoxin FBS". We have now compared this FBS to at least 4 other batches from different suppliers and have sufficient evidence to go back to the company in the new year...
It is known that the endotoxin level in fetal calf serum varies. It mostly depends on the company, they know in every case how "good" the fcs lot is they sell. Some companies mix the different FCS lots until it can be used for most cell lines. That's the way they can make a lot of money.
During this summer a company (is it allowed to tell you the name?) diluted the fcs with water and added some growth factors to compensate the bad quality. Some of my collegues had used this FCS because it was cheap and their cell cultures died. The whole story came up when GE Healthcare wanted to buy this company.
The only way to avoid such a trouble is, to test different lots. Use at least 5 of the most difficult cell lines you have and test these lines for at least 4 weeks. Usually I buy 100 bottles and store it at -80.
Good luck!
Hello Andrew,
I never see this type of problem but probably your FCS is contaminated by toxins or bacterias. In all my experiment (24 H treatment) I didn't used FCS and cells are growing well with a very low H2AX basal phosphorylation.
Good luck.
Marc
I agree with the previous answers. test different serums before use in rutine culture.
Especially if you are working with fanconi.
Silke, are you referring to the sera which prompted the following 'product information' statements?
"These products may contain added adult Bovine Serum Albumin (BSA) of United States origin, water, and/or cell growth promoting additives. All other FBS products... may contain adult BSA of United States origin, water, and/or cell growth promoting additives."
"The 48% supplement mix that we add ... is detailed below.
•31.5 g/L Bovine Serum Albumin (BSA) that is sourced from Proliant in the U.S.
•30.25 g/L Soy Peptone (Hy Pep 1510) that is sourced from Kerry Laboratories and manufactured from soy beans in the U.S.
•3.8 g/L Sodium Chloride that is sourced from Salinen Austria Group and manufactured from mineral salt.
•1 g/L glucose (Dextrose) that is sourced from Lobingerchemie. This component is manufactured in France by hydrolysis of plant-derived starch."
Is this just a case of the FBS being watered-down so that they can sell more volume?
Are there actual *growth factors* added to the sera you are referring to, or are the 'growth promoting additives' just glucose etc.?
re: the comment above, DNase is a very unstable, inefficient enzyme, and not likely to be the cause of your problems.
How are you detecting DNA damage? There are several possible things that can cause high background in some DNA damage assays, and it's probably not the serum unless it's just sticking to the cell and cross-reacting with your detection agents, e.g. if you are using antibodies.
Other things to consider: have you ruled out that it's the plastic dishes? There's good evidence of extracellular matrix signaling to DNA damage kinases, so if you're using some kind of IF or other phosphorylation as your readout, that could be part of the problem. When I ran into this issue, I was able to mitigate the effects by using fibronectin on the dishes (see my publications, website, or contact me if you'd like protocol details).
If you really think it's the serum, have you tried filtering it or heat-inactivating it? have you compared with serum from researchers in nearby labs?
I've also seen some strange side-effects from drugs that seemed to react with cell culture media, so it's really important to do untreated controls as well as solvent-alone controls if you're treating cells with anything. DMSO can by itself stress the cells out, even at amounts lower than urban legend would have you believe were tolerable. In some assays, this will appear to be DNA damage, but as Claudia mentioned above, it can be hard to distinguish different kinds of toxicity. A lot of DNA damage markers light up when cells start to die and initiate apoptosis.
Yes, it has been shown in my research as: protective effect of nigella sativa and thymoquinone on serum/glucose deprivation-induced dna damage in pc12 cells, please see it.
It has been shown that serum/glucose deprivation (SGD) induces DNA damage in neurons as PC12 cells, therefore quality of FBS will be effective in this process.
DNAse activity can be assessed with radio-labelled DNA looking for non-preciptated counts against controls. See Perkin elmer web page for assistance in labelling:
http://www.perkinelmer.com/Resources/TechnicalResources/ApplicationSupportKnowledgebase/radiometric/dna_rna.xhtml
Or Enzo's fluor labeling kits if you wish not to work with radioactive materials.:
http://www.enzolifesciences.com/product-listing/?product_subtype=Labeling%20kit&text=DNA&gclid=CMHTlpO64LsCFU_NOgod3T8Aag
DNAse controls can be qcquired fomr Life Technologies:
http://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/dnase-i-demystified.html
One can nick translate Salmon sperm or lambda DNA with either fluoro or radio labeling and test against your FBS for activity.
primary fibroblasts have different plating efficienciesin different batches of FBS and lines with defects in DSB repair are often more sensitive to bad serum batches. sounds like there is some agent that induces damage at the replication fork in your case. We routinely batch FBS before purchase (testing for cloning efficiency of primary fibroblasts) and order a large batch (but pay as we use it). This has worked well for us and its best to minimise changes of batches by purchasing a lot
Hi, I am not sure what is the cause but I did test of apoptosis and used FBS to sustain the the cell growth and we are using in the lab. heat inactivated FBS you can buy or do yourself at the lab. heating the FBS. May be that solve your problem.
Hi we have faced ES cell (CGR8) death and the most prominent suspect is FBS (Hyclone).
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