Hello everybody, Currently I am in charge of the extraction of the extracellular matrix from the brain peremchyma of mice in order to allow the primary culture of tumor cells. This area of research is new for me. I have already obtained decellularized samples (whitish appearance, greatly reduced quantity of proteins and nucleic acids, etc.). Now, I'm trying to make a gel but I didn't succeed. I freeze-dried the samples and tried to digest them with a pepsin solution (10 mg of sample for 1ml of pepsin at 1mg/ml in Hcl) as indicated in scientific publication.
I don't know if this is normal but if we let the solution obtained sit for a few minutes, we can see 2 phases : a transparent phase and another more whitish but which is not hard and which resuspends very well when we shake lightly the tube. After balancing the osmotic force (with DMEM 10X) neutralizing pH (with NaOH), I tried to gelificate samples at 37°C (without prior dilution) but this did not work, the solution remained liquid. I don't know where the problem comes from. Did somebody have encountered this kind of problem? Do you have any advice to give me? Thank you in advance for your help. Valérie
Omair A. Mohiuddin I have leaved my sample 48 hours in pepsin/Hcl. I have tryied at 37°C, 60°C and room temperature. After pepsin digestion step, the solution obtained is never clear, there are always things in suspension...
If a tissue is not too rich in ECM, it is difficult to cross-link the digested ECM. Some crosslinkers, like EDC, may be required to polymerize it.
Digestion should be done at room temperature, to get a partial digest that gels later. You can try a higher concentration of pepsin (e.g. 2mg per 10 mg tissue) to better digest the tissue. Moreover, tissue digestion at 10mg/mL is lower end of the range to make a gel you might want to increase the amount of tissue. I haven't worked with brain, but in my experience of working with adipose tissue which is also an oily tissue I had to go up to 40-50mg/mL for appropriate gelation. In my experience, most tissues don't digest completely there will always be some undigested particles.
Omair A. Mohiuddin Have you already quantitate sGAG in your samples (Blyscan assay)? What values did you get? Thanks a lot
We have done sGAG quantification, but that depends on the type of tissue you're working with. I haven't worked with brain.
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