From: Bradley Bone (Bone.Bradley@mayo.edu)
Date: Thu Apr 09 1998 - 08:30:05 EST
I know that this is off topic, but it might be an issue that many of you should be interested in, especially if you perform "flow" experiments on in vitro cell lines. Once a cell line becomes contaminated with Mycoplasma, I have seen dramatic differences, by FACS of the expression of some cell surface markers, so I will answer Henry's question and hope that it might help others out. I routinely run assays on various cultures to detect mycoplasma. I have extensively used the GenProbe Mycoplasma Rapid Detection System and it works very well. It runs $344 for 20 assays in the Fisher Catalog so it might be a little expensive. It is very sensitive and rapid and have not had any problems with inconclusive test results. Another test which I have used a lot is Gibco BRL's MycoTect Kit which is significantly cheaper ($136 for 50 assays). You do need to also have the indicator line which is the ATCC cell line (CCL96) 3T6 cells. This test takes 4-5 days to get the results. Generally, it works quite well, but sometimes the results can be inconclusive. There are a number of other methods used to detect mycoplasma including PCR, culture and fluorescence microscopy using dyes such as DAPI, that specifically stain double-stranded DNA, but I don't have extensively tried these methods. As far as cleaning up contaminated lines, contaminated lines can either be passaged through mice (only on certain mouse cel lines) or Boehringer Mannheim sells an antibotic combination (BM-Cyclin) which can be tried. >From a single experience using this antibiotic, I found that the procedure can take quite a while and depending on the cell line, the doubling rate of your cells can really go down. If it is a real valuable line that cannot be reaquired easily, I would give the antibiotics a try, otherwise I would try to reaquire a clean line. Regards, Bradley At 02:51 PM 4/8/98 -0500, hwortis_sup@opal.tufts.edu wrote: > >I need some help regarding tissue culture and thought that flowers might >have the experience I lack, or will be able to direct me to those that do. > >My lab has identified mycoplasma in one of our cultures. We had avoided >this for many years by careful monitoring of all new cell lines and by >several cautionary steps. > >My questions are: > >1. Which of the available Mycoplasma detection techniques do you prefer >to use? > >2. Do any of you have a preferred treatment method? (I know that there >are several relatively new drugs that are available.) > >3. Is there a site or source that I should check out for more >information? > >Thanks in advance, > >Henry H. Wortis >Department of Pathology >Tufts University School of Medicine >136 Harrison Ave. >Boston MA 02111-1800 >(617)-636-6718 >FAX (617)-636-8590 ------------------------------------ Bradley Bone Immunology Core Facility Coordinator Mayo Clinic Scottsdale Johnson Research Building 13400 E. Shea Blvd. Scottsdale, AZ 85259 Phone: (602)301-7131 Pager: (602)301-5753 FAX: (602)301-7017 E-mail: bone.bradley@mayo.edu website: http://www-sdl/research/imm/coreimm.html
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