I'm cultivating 3T3-F442A murine pre-adipocytes for an experiment. They suddenly started growing slowly and showed abnormal morphology.
I suspected of mycoplasm and as I don't have a kit for testing this infecting I did a hoechst staining after fixating my cells with 1:3 glacial acetic acid : methanol.
I only had acess to a 200X magnification which isn't ideal, but I could see dots on the cytoplasm.
I have annexed the images, are my cells infected or the dots could be mitochondria or artifacts?
ps. I have posted photos of 2 wells with two different exposure times, when I expose enough to see the dots on the cytoplasm the nucleus is too bright.
Your picture is way over exposed. Mycoplasma, when present, are visible even with a "normal" exposition. To be able to compare you could include a non infected cell line as reference. What strikes me, is the very homogeneous distribution (soup) of the cytoplasmic signal; usually micoplasma form rather coarse distinct dots. Moreover, on the picture taken with a shorter exposition time no cytoplasmic signal is seen. What you see could well be an artifact due to over exposition.
When cells get old (lots of passaging), they will also tend naturally to grow slower and get more difficult to transfect. Your problem may not necessarily be micoplasma...although it could.
Thiago,
I use to work with fluorescent dyes in living cells and I can tell you that healthy mitochondria should look like a fibers around nucleus instead of dots. On the other hand, stressed mitochondria could render a punctuated pattern but still you have these dots at the cellular border, so for me these correspond to mycoplasm. It makes sense with slow proliferation and morphologic change you describe as well.
Regards, OP.
Your picture is way over exposed. Mycoplasma, when present, are visible even with a "normal" exposition. To be able to compare you could include a non infected cell line as reference. What strikes me, is the very homogeneous distribution (soup) of the cytoplasmic signal; usually micoplasma form rather coarse distinct dots. Moreover, on the picture taken with a shorter exposition time no cytoplasmic signal is seen. What you see could well be an artifact due to over exposition.
When cells get old (lots of passaging), they will also tend naturally to grow slower and get more difficult to transfect. Your problem may not necessarily be micoplasma...although it could.
Usually mycoplasma is quite well visible with a normal exposure, with nucleus well visible with clear borders and the additional staining from mycoplasma more concentrated at the cell borders, as mycoplasma likes to be connected with cell membrane. However, in this case two photographs are overexposed and two underexposed, so I cannot say. The best it is to look directly under the microscope and when you see nucleus clearly, then look for mycoplasma.
As say Cyrille your picture is and mycoplasma, when present, are visible with a normal exposition. I recommended you PCR testing to be sure that it is mycoplama. I think is better do not treat the cells with plasmocin if you can use a new aliquot of the cells.
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