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Isolation of murine intestinal subepithelial myofibroblasts


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#1 Myofibroblast

Myofibroblast

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Posted 22 October 2013 - 03:56 AM

Dear all, 

 

I am a master student working in an immunology lab at a hospital on my thesis, which is to isolate, culture and characterize murine iSEMF. The isolation is working quite nicely - 2 times enzymatic digestion with colagenase XI and dispase for 10 and 30min, the 2nd fraction is then taken into cell culture. The cells are analyzed with FACS and qRT-PCR and there is 60-70% of aSMA+ cells after 1st passage and 75-80% of aSMA+ cells (out of lin.negative, when gating on EPCAM and CD31) after passage 2.

 

The 1st problem I am facing at the moment is the fact that my boss wants higher purity of the iSEMF culture. Do you have any ideas or hints how to achieve it? I am the only one in the lab working on those cells and could not find any publication stating this issue. Sadly he does not allow me to order vimentin, so I am working with desmin (cells are negative), aSMA (cells are positive) and Thy1.2 (cells are positive in 10-20%). 

 

my 2nd problem is a fungal contamination - a mold fungus, that is coming even I have 1x Amphotericyn B in my DMEM. When changing the medium every day, there is no aparent contamination. However when I leave it for 3 days or longer, first colonies can be recognized. I then wash the flasks several times with PBS + 2x Amph.B and change the medium. The cells are growing without taking any notice, but the contamination is annoying and I assume the fungi secrete toxic compounds into the medium, that might affect the cells.

 

I am washing the tissue after resection several times and am used to work sterile in the hood. 

 

Is there a way how to reduce / kill off the fungi?

 

Thanks a lot!






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