What is the best possibility to extract cells out of 3D bone replacement matrix (Bio-Oss) in an in-vitro project in order to measure the proteomics via mass spectrometry?
What do you think about grinding the matrix and using the "Pierce Mass Spec Sample Prep"-Kit for sample preparation?
An alternative method could be bone decalcification via EDTA and the use of the "Pierce Mass Spec Sample Prep"- Kit.
The samples were fixed on paraformaldehyde and are currently frozen at - 80°C.
Thanks a lot
All the best
Hello, dear Daniel! I am a maxillofacial surgeon who deals with reparative regeneration of facial skull bones. My experience with samples of lower jaw bone fragments has shown that the use of EDTA gives better results for studying the spatial organization of the fibre skeleton of the bone and cellular elements. I fix the material in a 4% solution of paraformaldehyde on O,1 M phosphate buffer (pH 7.2-7.4) containing 7.5% sucrose (O,3 osmomol). They were dehydrated in solutions of acetone of ascending concentration and dried by the method of crossing the critical point on the NSR – 2 device (Hitachi). I decalcify in EDTA at a temperature of 37 degrees C. I do not know how this scheme will work with the tissues that you have frozen. I wish you good luck and good health in this challenging time.
EDTA is well established for this purpose and does work, but for increased efficiency we moved to a 4% Formic acid solution at room temperature, which is typically that same acid and concentration used in proteomics for stabilizing the solute. Then you perform your typical extractions.
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