I am working with HTC75 cells (human fibrosarcoma). I am new to IF and I am not sure if these DAPI staining pictures of the cells are normal.
The cells were not treated with anything.
Growth media: 1x DMEM, 10% BCS, 1% penn-strep, 1% L-glutamine
The cells were grown on coverslips until 70% confluency.
Fixed with: 3% paraformaldehyde/ 2% sucrose
permeabilized with Triton-X buffer:
0.5% Triton X-100
20mM Hepes-KOH (pH7.9)
50mM NaCl
3mM MgCl2
300mM sucrose
Stained for DAPI for 1 minute and washed with 1x PBS for 5 minutes.
Would anyone please tell me if these cells are contaminated? The pictures' qualities are poor because the software automatically adjusts the contrasts of the pictures, so I took them with my phone.
The cells looks to me as normal without any contamination. Generally, either mycoplasma, fungus or bacteria contamination may be found in this kind of cell culture. When you are looking through inverted microscope the mycoplasma contamination may be looked like black small spot in the cell cytoplasm. Regarding contamination with bacteria or fungus the transparency as well as pH and color of the medium may be changed (off note, the pink color of medium is because of phenol red which is a pH indicator).
Difficult to see from your pics, but you may have mycoplasma contamination (DAPI-positive dots in the cytoplasm). Usually when it's visible with DAPI, it suggests that they are contaminated pretty heavily . More sensitive kits are available that allow you to ascertain / measure it, either based on light-emitting reagents (easy) or PCR (sensitive!).
Mycoplasma contamination may also affect the proliferation of your cells and your ability to transfect them, so it's always a good idea to check routinely in the lab.
As a rule DAPI staining of extranuclear DNA present in mitochondria is imperceptible under normal fluorescence microscope and thus any extranuclear bright spots indicate presence of mycoplasma. In your case presence of extranuclear DNA in first micrograph do point towards mycoplasma contamination. You should discard these cells and media and sterilize both incubator and cell culture facility . Prepare everything fresh and see if the problem persist. In case you can not discard the cells then you can go for mycoplasma treatment by using ciprofloxacin (10 microgram/mL) as per the attached article. I have treated one of my cell line using same procedure.
http://www.ncbi.nlm.nih.gov/pubmed/3282011
I agree with previously mentioned comments. Its hard to say from the images but in the first image I can clearly see extranuclear staining suggesting presence of mycoplasma. If that's how your culture looks over all, then you will have to get rid of them. Any transfections, signalling, proliferation tests done on myco-contaminated cell lines will give you skewed results, even though the cells seem to look alright. Goodluck!!
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