MG63 is an osteosarcoma cell line which is an accepted model of osteoblasts. My sample is a bone graft material in powder form. Using ionic dissolution method I used to treat it on MG63 cell lines. So far I have known that it is the only cell line with the characterization of osteocalcin secretion, and another try on SaOS-2 (another osteosarcoma cell line) was also a failure. There were literature saying that in response to 1,25-dihydroxy vitamin D3, secretion of osteocalcin was found to increase in MG63 cell line. I purchased it from Cayman Chemical and did a trial as per that research paper but it was not successful. I have modified cell density & concentration of vitamin D3 but not sure of the origin of the problem. Kindly help in the troubleshooting. Thank you.
How are you measuring your OC? If it is ELISA then you will have issues because the OC protein will be, for the most part, attached to the extracellular matrix. I suggest immunoblotting or real time PCR. Not ideal, but it should work for you.
Jenifa,
I agree with Carl especially with ensuring that the cells make OCN by measuring BGLAP (OCN gene) mRNA levels using PCR. If you do not detect high mRNA, then there is no point of trying ELISA. Also, many osteoblast-like cells including MG63 have lost their differentiation potential and no longer behave like authentic primary osteoblasts. Finally, there are carboxylated and uncarboxylated forms of osteocalcin protein.
Carl Gregory Thank you for answering. Yes, I have used ELISA method only. Having doubt on OC protein being attached to extracellular matrix, I lysed the cells and analysed both intracellular and extracellular contents of MG63 cells. I will take your suggestion of going for immunoblotting or real time PCR.
Hesham A. Tawfeek Thank you for your answer. In such case of MG63 losing its characteristics of authentic primary osteoblast, what could be the best option to choose another human cell line with such characteristics. Could you help on that please.
Try first measuring BGLAP mRNA in MG63 using PCR or RT-PCR. If you detect several fold increase in expression, then I would try, as Carl suggested, testing the whole cell and matrix lysate. If you do not see reasonable mRNA expression, then it is unlikely that you will detect any OCN protein.
You can always isolate primary osteoblasts from bone marrow by culturing in osteogenic medium and use them to detect OCN protein. However, I would always first ensure that the mRNA levels are high enough before looking for the OCN protein.
With all of that said, OCN ELISA kits in the market are not the best and you have to keep in mind whether your kit detects whole, carboxylated, or uncarboxylated OCN. Also, check to see if the ELISA kit was ever tested for measuring cellular OCN. Most kits have been optimized and tested for serum.
I would take a look by immunoblotting because OC is a sticky protein, but as mentioned, MG63 cells are not ideal tools for studying osteogenesis. I suggest mesenchymal stem cells, but I am biased because I work on them!
Carl Gregory Thank you very much. I would take that suggestion. Do you work on mesenchymal stem cells?
Yes, my group has worked on them since 2005. I have written a few protocols on general culture, many specializing in bone assays.
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