I am maintaining the LNCaP cells. I am observing black dots in background medium. Initially, the dots were less in no. but they increased after subsequent passages and now my cells are having problem in adhesion to the culture plate and they are not looking healthy. I did DAPI staining to identify the problem, I got the staining similar to mycoplasma contamination. So, I terminated the culture and tried to revive fresh vial but I was unable to revive as my cells did not attach to the plate and the plate was full of those black dots. Next, I took cells from one of my labmate who is maintaining the same cells, but not having this problem. His cells were healthy. I tried to maintain them, but still getting those black dotes. So, I took media from my labmate who gave me cells. With his media, the problem of dots decreased. Thus I thought there is problem in my media, but every time, I prepared media, I was having the same problem. So, can somebody tell me what are those dots? And how can I get rid off these dots.
Hi Lalita, don't waste your time trying to eliminate the infection in your cell culture. It will not work. Through them into the autoclave. Also take care of any infection in the cell culture hood, incubator, etc. Starting a new batch of cell will not help if you did not take care of any area that is in prolonged contact with the cells (see also http://www.bionique.com/mycoplasma-resources/faq/mycoplasma-enter-cell-cultures.html; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463982/pdf/10616_2004_Article_5113481.pdf). There are lots of inexpensive tests out like from Lonza (MycoAlert™ PLUS Mycoplasma Detection Kit), Invivogen (PlasmoTest™), lifetechnologies (MycoFluor™ Mycoplasma Detection Kit). Hope that helps.
Lalitha, I think the problem comes from the serum that you are using and not the media. Have you heat inactivated the serum before use? Serum may be a source of mycoplasma contamination. You can also filter the media and serum using sterile syringe filters just before culturing cells. We follow the same procedure in our lab. Moreover, you can add plasmocin to the media or can use a mycoplasma spray in the hood (if available).
Sometimes, the black dots may not be mycoplasma. You can try discarding the trypsin that you add to the culture flask during subculturing (be careful not to lose cells along with trypsin removal). (Remove trypsin after allowing the cells to shrink. Shrinking helps the cells to get easily detached from the flask by adding media forcefully into the flask.) Usually we add 10% serum -containing medium directly to the trypsin present in culture flask for inactivation of trypsin, right? I suggest to remove trypsin first and then add 10% serum -containing medium. Then pelleting can be carried out. This procedure removes those contaminants to a significant degree. Anyway you need to standardize the time of incubation with trypsin because it varies with different cell types.
It does sound a lot like mycoplasma, given the DAPI staining, though of course we can't be certain.
Mycoplasma is quite an insidious form of contamination, usually linked to operator technique. Once contamination is present, getting rid of it is hard if you don't eliminate the source(s). I recommend reading the following review paper for more information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584481/
I would toss all reagents that you have been using, as well as the cells. Next, implement a regular testing regimen for mycoplasma, it will be worth it. If you have a lab mate without this problem, you're blessed that the contamination hasn't really taken root in your cell culture facility.
Hi Lalita, I had that problem some time ago with a cell line, the best is to throw your opened reagents away, a little bit drastic but effective solution since is an insidious contamination. If is not possible, I treated my cells with the "mycoplasma removal agent" from AbD Serotec and worked great :)
I agree with Peter and Maria that you probably should throw away all opened reagents. And additionally clean very properly incubator where the contaminated cells were.
PCR to detect the mycoplasma contamination followed by treating cell line with mycoplasma removal agent (MRA) should solve the problem.
PCR is a reliable, fast, and sensitive method and should be applied regularly to monitor the contamination status of cell cultures.
MRA is non toxic, and will not interfere with the viability or function of cells in culture. It should be emphasised that MRA should not be used as a substitute for good cell culture techniques.
Refer: PMID: 21516400. Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. Methods Mol Biol. 2011
Hi Lalita, don't waste your time trying to eliminate the infection in your cell culture. It will not work. Through them into the autoclave. Also take care of any infection in the cell culture hood, incubator, etc. Starting a new batch of cell will not help if you did not take care of any area that is in prolonged contact with the cells (see also http://www.bionique.com/mycoplasma-resources/faq/mycoplasma-enter-cell-cultures.html; http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463982/pdf/10616_2004_Article_5113481.pdf). There are lots of inexpensive tests out like from Lonza (MycoAlert™ PLUS Mycoplasma Detection Kit), Invivogen (PlasmoTest™), lifetechnologies (MycoFluor™ Mycoplasma Detection Kit). Hope that helps.
My advice is to discard your available cell lines and already prepared media.
Secondly clean your incubator and biosafety cabinet with 70% IPA. If it is not work fumigate the equipment using formalin and KMnO4 ( Adding 35 mL of formalin (40 percent formaldehyde) to 10 g potassium permanganate per cubic metre of space) But some people don't recommend the fumigation
Thirdly prepare the new solutions and filter through sterile syringe filter (especially you have to filter after adding the serum, because most of time serum would be the contaminant)
Use a proper antibiotic mixture. I use gentamycin 50ug/ml and amphotericin 1ug/ml solution
Thank you all of you. I cannot remove trypsin before collecting cells as my cells are v. loosely adherent. they will come along the trypsin.
detect mycoplasma contamitation via PCR of the supernatant. Primer seqs are available online.
To treat the culture use the Roche BM Cyclin Kit. This worked great in my hands.
All the best
Except all these mentioned above you should check also the conditions using by others in the same lab and the situation of their cultures. Maybe is coming from other cultures using the same incubator.
If the cells you are working with is very important and difficult to get the new batch, the information from this paper might give you the idea how to treat mycoplasma contamination.
PLoS One. 2013 Jul 30;8(7):e70267. doi: 10.1371/journal.pone.0070267. Print 2013.
Effect of antibiotics against Mycoplasma sp. on human embryonic stem cells undifferentiated status, pluripotency, cell viability and growth
However, I do agree with all suggestions above.
Filter all media containing both FBS and antibiotic/antimycotic with a .22 PES filter. Do it in a hood. Use ATCC's Universal Mycoplasma Detection Kit to confirm presence of mycoplasma. Also mycoplasma are typically not visible with an inverted brightfield scope. You would need an EM to see them. Sounds like you might have a bacterial infection, in my opinion.
suggest Gibco's Antibiotic-Antimycotic (100X) at 1% in media. http://www.lifetechnologies.com/order/catalog/product/15240062
Has anybody used the kit from Biotools? It's a colorimetric assay, very fast (10min), but I am not sure about its specificity. We also used the Venor GeM kit from sigma aldrich, but everything shows up negative, and the way to isolate the DNA is by boiling the supernatant, has anybody experienced this PCR kit?
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating