Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Sergio.
We digest chondro pellets using papain and then proceed to the DMMB assay. Protocol below;
3.1.1 Weigh out 1mg of papain and dissolve in 10ml of dilution buffer (may dissolve in weigh boat as papain is fine powder)
3.1.2 Rinse weigh boat repeatedly to ensure all papain is transferred
3.1.3 Invert several times to ensure even distribution
3.1.4 Add 250µl of this solution to 9.75ml of dilution buffer
3.1.5 To each pellet add 200µl of the diluted papain and digest overnight at 60ºC
3.1.6 Vortex samples to ensure pellet is dispersed
Papain Sigma P4762
Also this pellet will be digested so you rightly stated it will not be suitable for histology.
Good luck!
You could use this buffer : the samples were digested in 0.1% papain
#0.1% proteinase K in 1 mM CaCl2, 10mM Tris HCl, pH 8 at 60C for 24 h with low stirring.
I have published this protocol here....
Article Native and DPPA cross-linked collagen sponges seeded with fe...
Hi Benjamin. You can use the DMMB dilution buffer you will be using for the DMMB assay itself.
I would suggest a slightly different technology if you would like to have quantification, visual identification, and standardization to DNA content. We developed a procedure called an ELICA (enzyme-linked immuno-culture assay) that can be adapted to any situation (we routinely used it in wells of 96-well plates, on cryosectioned material, etc), but you will need access to UV/Vis spectrophotometer and bright field microscope. In your case, if you are looking at cell pellets I would suggest cryosectioning for tissue sections first. Our fixative of choice for retention of "everything" was a combination of glutaraldehyde (2.5%), formaldehyde (4%), D-glucose (1%) in DPBS. Morphology would be retained as well as antigenic sites. Use antibody to proteoglycan of choice, containing GAG side chains (antibodies can be acquired from Developmental Studies Hybridoma Bank, University of Iowa). Bind antibody to section. Then anti-species antibody with biotin attached, then avidin-HRP (basically 10x amplification procedure). Then incubate with soluble HRP substrate, remove and read in Vis-spectrophotometer. Wash out residual soluble HRP substrate and incubate with insoluble HRP substrate - photograph. The hydrolyze section with acid and perform DNA analysis. Therefore your results will be standardized to the number of nuclei (cells) in your sample. Results were nanogram quantities of phenotypic expression marker per microgram DNA. Protocols, reagents, etc.can be found in two articles:
Young HE, Sippel J, Putnam LS, Lucas PA, Morrison DC. Enzyme-linked immuno-culture assay. J Tiss Cult Meth, 14:31-36, 1992.
Young HE, Duplaa C, Romero-Ramos M, Chesselet M-F, Vourc’h P, Yost MJ, Ericson K, Terracio L, Asahara T, Masuda H, Tamura-Ninomiya S, Detmer K, Bray RA, Steele TA, Hixson D, El-Kalay M, Tobin BW, Russ RD, Horst MN, Floyd JA, Henson NL, Hawkins KC, Groom J, Parikh A, Blake L, Bland LJ, Thompson AJ, Kirincich A, Moreau C, Hudson J, Bowyer III FP, Lin TJ, Black Jr AC. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys, 40(1):1-80, 2004.
Alternatively, you could use an Alcian blue based assay. But be forewarned they stopped making Alcian blue back in 1979 due to its highly carcinogenic activities during manufacture (large number of employees making stain were getting cancers). So any Alcain blue purchased after 1979 is not anywhere near to being 100% stain, it is mostly fillers. Alcian blue assay protocols can be found in:
Young HE, Dalley BK, Markwald RR. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.
Young HE, Young VE, Caplan AI. Comparison of fixatives for maximal retention of glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S]sulfate. Journal of Histochemistry and Cytochemistry, 37:223-228, 1989.
Young HE, Carrino DA, Caplan AI. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.
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