Which viability assays can I do to confirm chondrogenic differentiation from MSC pellets? Cells were pelleted in micro centrifuge tubes and differentiated.
I would agree with Dr. La Rocca, Alican blue at pH 1.0 can distinguish sulfated glycosaminoglycans (GAGs), whereas Alcian blue at pH 2.5 can distinguish carboxylated GAGs. One could also use Safranin-O. It is another stain that will distinguish sulfated and carboxylated GAGs at similar pHs. Combining staining with Alcian blue at pH 2.5 followed by Safranin-O at pH 1.0 one can distinguish different GAGs within the same tissue section. Blue staining only will identify GAGs having only carboxylated groups, such as hyaluronic acid. Orange staining only will identify GAGs with only sufated groups, such as keratan sulfate and heparan sulfate. Whereas, a brown/purple stain will identify GAGs with both carboxylated (blue) and sulfated (orange) groups such as chondroitin sulfate and dermatan sulfate. The predominant proteoglycan (PG) in cartilage is Aggrecan which is a collection of PG monomers containing both chondroitin sulfate GAGs and keratan sulfate GAGs. The monomers are held together with a long hyaluronic acid molecule. There are also individual keratan sulfate PGs and chrondroitin sulfate PGs in cartilage, as well as the predominant type-II collagen. Type-IX collagen is the bridge molecule between type-II collagen and the PGs. The antibodies for type-II and type-IX collagen can be acquired from Developmental Studies Hybridoma Bank at the University of Iowa. The bank is subsidized by NIH, although anyone anywhere in the world can purchase from the bank. The best company to purchase stains, in my opinion, is Chroma-Gesellschaft.
Hello
They are nearly the same as you differentiate cells in a three-dimensional hydrogel: Alcian blue stain versus control cells (in this case in my experience only differentiated cells form an extracellular matrix which is robust enough to survive fixation and inclusion in paraffin), collagen II expression, other cartilage markers (COMP, collagen X, etc.), both at mRNA and protein level.
There are other stains/markers applicable, but these constitute a good basis to start with.
Hope this helps.
If you want maintain alive your cells, you could try to detect collagen II or X in the culture medium at the protein level. It is challenging due to the amount of protein.
You could try to pool culture medium, concentrate and do WB. If you have a antibody against the triple helix of collagen II, you could perform a acidic pepsin digestion that remove all proteins and globular part of collagen.
I would agree with Dr. La Rocca, Alican blue at pH 1.0 can distinguish sulfated glycosaminoglycans (GAGs), whereas Alcian blue at pH 2.5 can distinguish carboxylated GAGs. One could also use Safranin-O. It is another stain that will distinguish sulfated and carboxylated GAGs at similar pHs. Combining staining with Alcian blue at pH 2.5 followed by Safranin-O at pH 1.0 one can distinguish different GAGs within the same tissue section. Blue staining only will identify GAGs having only carboxylated groups, such as hyaluronic acid. Orange staining only will identify GAGs with only sufated groups, such as keratan sulfate and heparan sulfate. Whereas, a brown/purple stain will identify GAGs with both carboxylated (blue) and sulfated (orange) groups such as chondroitin sulfate and dermatan sulfate. The predominant proteoglycan (PG) in cartilage is Aggrecan which is a collection of PG monomers containing both chondroitin sulfate GAGs and keratan sulfate GAGs. The monomers are held together with a long hyaluronic acid molecule. There are also individual keratan sulfate PGs and chrondroitin sulfate PGs in cartilage, as well as the predominant type-II collagen. Type-IX collagen is the bridge molecule between type-II collagen and the PGs. The antibodies for type-II and type-IX collagen can be acquired from Developmental Studies Hybridoma Bank at the University of Iowa. The bank is subsidized by NIH, although anyone anywhere in the world can purchase from the bank. The best company to purchase stains, in my opinion, is Chroma-Gesellschaft.
If you really need viability assays in this case the choice will depend on the data you need to get : is it metabolic health/status of cells, or general amount of proteins they produce, or just a number of cells per pellet, or may be DNA degradation/apoptosis?
Thanks for the answers everyone. Trying to find a more quantitative method, so the expression of collagens is great and i have attempted it previously but with mixed results. What are your thoughts on looking at 35S incorporation?
Sorry, Yuriy, twitchy fingers, i did mean viable assays.
35S incorporation will need to be addressed with respect to the amount of CS-PG, KS-PG, and CS/KS-PG in your chondrocytes. With the ability of the KS molecule to structure up to 5 sulfate molecules (it is variable dependent on age of the cell) it will be interesting to quantify. Alternatively, you might try 14C-glucosamine. It is more of a 1:1 with respect to the ECM components, and the half life is much longer, so you can use significantly less of the isotope for your assays.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques