Hi Martin, I am sorry for you. You have just faced the nightmare of every tissue culture person in the world. I met mycoplasma more than 30 years ago, when I was ready to publish one article and my experiments stopped to reproduce results since mycoplasma "eats" nucleotides, degrading them to nucleosides (i.e Thymidine to thymine).

Whatever you try and in spite of the vendors that claim so, mycoplasma will come back (because it never has gone) if you do not change habits in your tissue culture lab. But in order not to be so negative I would recommend some tips to reduce the amount and continue working under a control undetectable level with them.

1) All the reagents recommended above will be suitable. Some more than others. I tried at least 4 of them including Mycozap from Lonza, cyprofloxacin to treat (20 ug/ml) and to maintain (5 ug/ml).

2) There is one "two steps kit" that was also very effective but do not remember the brand.

3) Before doing the treatment, keep the cells in media WITHOUT any antibiotic. This will prevent the mycoplasma to internalize and be protected from the antibiotic treatment.

4) You can also help by reducing the number of myco particles after culturing without antibiotic (such as P/S) for several days, by washing the cells quickly with tissue culture WATER (yes, you read it correctly). You can wash the cells with PBS after removing the media and before the water. Leave the water for 30- 45 seconds. The cells will resist, the myco will "explode".

5) Then you can replace the media for 1 day and the following day you will perform your myco specific treatment.

6) Optative: It may be useful to identify the strain you have. There is a small company in UK where I sent my cultures and they told me the name and the antibiotic sensitivity. I have to look on my notes for the address. It is affordable.

7) When everything above is done, or better, before is done, you have to use only pipets with cotton plug and a pipet aid to discard the media in a container before splitting your cells, AND NOT THE VACUUM PUMP TO ASPIRATE. THE VACUUM IS THE FAST WAY TO CONTAMINATE YOUR CULTURES, even if you change the aspirating pipet each time or if you clean with bleach the tubing between aspirations (which is more dangerous, since the liquid microscopically makes reflux inside the tubing and it is very easy for the myco to swim back and of course the bleach).

8) On a quote, I remember a laboratory in France that used to culture separate mycoplasma free and the same mycoplasma contaminated cell line because the myco induced the expression of the protein they were working with. Of course this was done in separate tissue culture rooms.

Good luck!

Hi,

Try this: http://www.invivogen.com/plasmocin

If it doesn't help, your contamination might be systemic.clean and scrub water Bath, incubators, etc...

Hi Martin, you could try Plasmocin from Invivogen. Works reasonably well in just a couple of weeks!

Hi Martin,

sorry to hear about that. I had similar problem in the past and I tried several reagents, but the best in our hands was MycoZap from Lonza (see link below)

Good luck!

(http://www.lonza.com/products-services/bio-research/cell-culture-products/mycoplasma-detection-and-removal/mycozap-mycoplasma-elimination-reagent.aspx).

Plasmocin from Invitrogen will be good!

I've always had good results with either plasmocin or ciproxin. Best to try at different concentrations as some cells don't cope well with the treatment.

I have used plasmocin in the past, with different ceel lines I had great results!

best of lucks with your cells!

The mycoplasma removal agent by AB Serotec is also a great prodcut to try. http://www.abdserotec.com/product/mycoplasma-removal-agent-buf035.html

Biomyc wiil be good option, I use it in the lab... Good Luck!

http://www.bioind.com/page_14007

Hi Martin,

we treat our cells with ciprofloxacin (20 ug/ml), from SIGMA (17850-5G-F) during 1-2 weeks,

Good luck

Thank you all for your advice!

The plasmocyne works well but in case of resistance I use plasmocure (see link below) which works very well on plasmocyne resistants (2 weeks additional cure)

http://www.invivogen.com/plasmocure

Gilles

Were treated cells selected after Plasmocin treatment? thanks

I suggest you treat with high concentration of mixed antibiotic for long time, of course after 2-3 weeks, part of the cells are also lost, but you get freed. If you have the source of your cells available, may be you take new sample and culture them with higher concentration than normal, e., double of 50 microgram or so.

Hi Martin, I am sorry for you. You have just faced the nightmare of every tissue culture person in the world. I met mycoplasma more than 30 years ago, when I was ready to publish one article and my experiments stopped to reproduce results since mycoplasma "eats" nucleotides, degrading them to nucleosides (i.e Thymidine to thymine).

Whatever you try and in spite of the vendors that claim so, mycoplasma will come back (because it never has gone) if you do not change habits in your tissue culture lab. But in order not to be so negative I would recommend some tips to reduce the amount and continue working under a control undetectable level with them.

1) All the reagents recommended above will be suitable. Some more than others. I tried at least 4 of them including Mycozap from Lonza, cyprofloxacin to treat (20 ug/ml) and to maintain (5 ug/ml).

2) There is one "two steps kit" that was also very effective but do not remember the brand.

3) Before doing the treatment, keep the cells in media WITHOUT any antibiotic. This will prevent the mycoplasma to internalize and be protected from the antibiotic treatment.

4) You can also help by reducing the number of myco particles after culturing without antibiotic (such as P/S) for several days, by washing the cells quickly with tissue culture WATER (yes, you read it correctly). You can wash the cells with PBS after removing the media and before the water. Leave the water for 30- 45 seconds. The cells will resist, the myco will "explode".

5) Then you can replace the media for 1 day and the following day you will perform your myco specific treatment.

6) Optative: It may be useful to identify the strain you have. There is a small company in UK where I sent my cultures and they told me the name and the antibiotic sensitivity. I have to look on my notes for the address. It is affordable.

7) When everything above is done, or better, before is done, you have to use only pipets with cotton plug and a pipet aid to discard the media in a container before splitting your cells, AND NOT THE VACUUM PUMP TO ASPIRATE. THE VACUUM IS THE FAST WAY TO CONTAMINATE YOUR CULTURES, even if you change the aspirating pipet each time or if you clean with bleach the tubing between aspirations (which is more dangerous, since the liquid microscopically makes reflux inside the tubing and it is very easy for the myco to swim back and of course the bleach).

8) On a quote, I remember a laboratory in France that used to culture separate mycoplasma free and the same mycoplasma contaminated cell line because the myco induced the expression of the protein they were working with. Of course this was done in separate tissue culture rooms.

Good luck!

Hi,

I recommend this;

http://www.mpbio.com/US/Pages/Product.aspx?pid=0930500

Good luck!

Hey Martin,

I have to agree with Laura Bover. You have just walked into a nightmare. After treatment your cells will change. That happened with us with some cells we have treated and then double checked for signaling pathways, morphology, behavior in vitro and in vivo. We used the kit from Sigma and Lonza when we had myco contamination.

We also checked the status via two different methods... a luminometer and PCR based kit. The PCR picks up very specific strains and the luminometer based kit picks up the more broad. Once both methods said negative we were sure they were myco negative. Good luck!

From my experience with mycoplasmas through more than 30 years, I agree with Dr. Hernandez. The use of ciprofloxacin is good sugestation.

Irina Grozdova Moscow State University

I used successfully Mycoplasma removing agent (MRA) from Invitrogen. I agree with Christian Regenbrecht that it may be helpful to check mycoplasma in the culture medium and steriilize the incubator.

Unfortunately Laura Bover's comments are true - you will never eliminate mycoplasma from your cultures. Mycoplasma also express high levels of ABC transporters so ANY drug studies with these cultures would be in error.

Hi Martin,

This is a good article that can provide you invaluable help:

http://www.jstor.org/discover/10.2307/4295311?uid=2129&uid=2&uid=70&uid=4&sid=21102049624391

Good luck!

Hi, we use BM Cyclin from Roche and we do not detect mycoplasma by PCR after 3 or 4 cycles with antibiotics. Maybe you have to treat your cells for a longer period of time.

Good luck, Inés

If your cell lines are very essential and especially if they are from patient origin, you may treat the cells with Cipro-bay for 2 weeks and then test them for mycoplasma. I am sure it will solve your problem. you may have to check the concentrations in the literature

Hi Martin, sorry to hear that:

On a good note now everybody knows that once your cells are carrying mycoplasma, you won't ever be able to get rid of it. What happens is that by treating them you lower the levels so, becomes undetectable and you think they are OK. If you keep passing them long enough without the treatment, it will be back. Also, I never liked to prepare experiments with cells that are growing under yet another set of chemicals that may affect pathways, etc.

I most definitively start all over re-establishing the cell lines....maybe they're available from ATCC/LONZA etc.

One very important thing I've always emphasized is to always use a lab coat you have dedicated to the cell cuture hood and gloves boxes next to it. Don't run around transforming bacteria and purifying plasmids and furthermore eating with the same lab coat. Always keep the different areas of lab work segregated with their own reagents, spaces, gloves, etc. GOOD LUCK !

An effective way to get rid of it is using Tylosin in every passage (or thawing, for new samples). Added to it, you should proceed a simple PCR to check if the culture is really clean.

For that, use the MGSO/GPO3 pair or primers. The positive reaction make a 270 bp band.

Mycoplasma is every scientist's nightmare! It has already led to many experiments being unpublished as the contamination will affect cell signalling and behaviour as already mentioned by others. We have had a massive outbreak about 2 years ago and eradicated this by cleaning all incubators, cabinets and tissue culture equipment. We discarded all contaminated cell lines unless this was not possible for which we then continued with ciprofloxin treatment for a few weeks. We test our cultures on a monthly basis using a PCR method to make sure that no contamination is present. Also all untested or new cell lines are put in a quarantine incubator and handled in a separate cabinet. Be aware that mycoplasma contamination can very easily spread between culture flasks and even within liquid nitrogen. Good luck!

When the BM cyclins don't work you it means that you may have another strain of mycoplasma. When this happens I normally treat cells for 15 days with Ciprofloxacin. You can get it from genHunter. Good luck.

Hi everyone;

Can I visibly detect Mycoplasma contamination in my culture, and what is the easiest way to investigate?

Hi Mohamed, we routinely use an ATP assay in conditioned media. The system is fast and very easy to use. You can also detect myco by staining your cells with Hoechst dye, but you need a trained eye to first recognize them.

Yehia, there is a PCR based assay from ROCHE...very sensitive against mostly all species.

I normally use MycoFluor reagent from Invitrogen to detect mycoplasma. Under the microscope it is visible as very bright and tiny blue sandcorns. I tried a PCR based method but ti can give false positives.

Beware of these microscopic techniques : they are not as sensitive as PCR, and you may think your infection is over while it's not, leading to the neverending story of mycoplasma.

Also, I would be curious to read any litterature on this long-heard story of contamination of vials in liquid nitrogen. I'm struggling to imagine this kind of thing.

@ Vincent...it is a nightmare as you would be surprised about the amount of different cell lines from different labs running all over the world sometimes generated in very prestigious labs and mycoplasma contaminated. But, NO ONE likes to document mycoplasma contamination because all published results and people may need to work with exact same cell lines, even if contaminated otherwise comes the question, "why can't you re-produce those results already published from such and such"...now you wonder...

Hi Martin. We use this product: http://www.abdserotec.com/catalog/antibody-manufacturing-tools/mycoplasma-removal-agent-mra.html

We usually get good results...

@Elda: I agree that mycoplasma is a big issue that probably affects the results.

Still, what are the odds that these contaminations occur in the N2 tank rather than in the incubator, the waterbath, the hood etc? Places were dilution is lower, temperature and agitation higher...

once in LN2 nothing will happen, if they are contaminated they'll keep the same...if not, they won't. This type of contamination occurs primarily when you establish the mother stocks via transduction with anything you choose to transform a primary cell culture into an stable cell line and mainly from FBS (must be screened...). And again I insist to respect the cell culture hoods and the surrounding areas as we are the only ones who bring cells in contact with anything and everything...

@Elda : OK, so you agree that LN2 is probably not a place where cross-contamination occurs. I misunderstood your previous post.

Hi Martin.You just mentioned about the use of ATP assay in conditioned media.Can you please elaborate about the assay or put up the link of some references for the same?

Hi Arnab,

we've used a variety of methods in our lab. Ultimately we were using this one:

http://www.biocenter.hu/pdf/Mycoalert.pdf

Also, there's a PCR-based detection method from Roche which is pretty good and more sensitive than ATP-based.

You can maybe use an ATP method as a routine method if you don't have usual mycoplasma contamination, and changing to PCR-based if you want, for example, to build a cell bank.

Hi Martin,

we use plasmocin.

I also think PCR is a sensitive and specific detection method because you will need to test the cells after your elimination procedures.

Combinations antibiotic should be good to use, like plasmocin which has both bacteriostatic and bacteriocidal antibiotics.

http://www.hindawi.com/journals/bmri/2012/267678/

This citation proposes and describes a detailed approach using a series of antibiotics for 21 days which may be helpful.

I agree that cell culture habits have to changed as the source is difficult to identify and mostly likely species found are human and bovine and spreads by droplet and thus source may be around the cell culture surrounding or from an infected cell line, which needs to be eliminated by combination antibiotic usage.

Hope this will help, best of luck!

Hi Martin. We use this product: http://www.abdserotec.com/catalog/antibody-manufacturing-tools/mycoplasma-removal-agent-mra.html

Same as above. Yout TC protocols must be strictly adhered too. I very rarely have mycoplasma contamination, unless it has come from elsewhere, as a vial or flask. When contamination is present I strip, clean, and autoclave every possible utensil that is used in my TC. I remove any cells and treat with Mycoplasama removal agent and place in a suitable incubator. Meanwhile I fully decontaminate my incubator, hood and water bath, including any racks etc present. I then test regularly my cells before splitting and keep treating with Removal Agent until clean. Regular Mycoplasma testing is essential. I always test my cells before injecting in vivo and before freezing down. I also re-check when revivng any cells. It is a total nightmare when cells are contaminated . I hope you get this problem resolved soon. Take care

Hi

You can use: http://www.abdserotec.com/catalog/antibody-manufacturing-tools/mycoplasma-removal-agent-mra.html

It will help you likely

Good luck