2.5 × 10⁵ BM-MSCs were cultured in a micromass pellet system as previously described [40 (link),41 (link)]. Pellets were cultured either in a growth factor free chondrogenic differentiation medium (CDM-) which consisted of high glucose DMEM supplemented with 1% penicillin-streptomycin, 100 μg/ml sodium pyruvate (Sigma), 40 μg/ml l -Proline (Sigma), 50 μg/ml l -ascorbic acid-2-phosphate (Sigma), 1.5 mg/ml bovine serum albumin (BSA-Sigma), 1× insulin transferrin selenium (ITS- Gibco), 100 nM dexamethasone (Sigma) for 28 days. This media was supplemented with 0.05 mg/ml of solubilised AC-ECM or LIG-ECM during twice weekly media changes. As a positive control, pellets were also cultured in CDM + that consists of the CDM formulation above plus 10 ng/ml transforming growth factor beta-3 (TGF-β3 –PeproTech). sGAG quantification was performed using a 1, 9 dimethylmethylene blue (DMMB) assay according to the manufactures protocol (Blyscan sulfated sGAG assay kit, Biocolor).
Blyscan sulfated sgag assay kit
Manufactured by Biocolor
Sourced in United Kingdom
The Blyscan sulfated sGAG assay kit is a laboratory equipment that quantifies the sulfated glycosaminoglycans (sGAGs) present in a sample. It provides a colorimetric method to determine the concentration of sGAGs in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β3, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid 2 phosphate, by Merck Group (1 mentions)
L proline, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Sodium phosphate, by Merck Group (1 mentions)
Quant it picogreen dsdna kit, by Thermo Fisher Scientific (1 mentions)
Na2edta, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
2 protocols using blyscan sulfated sgag assay kit
1
Chondrogenic Differentiation of BM-MSCs in ECM-Supplemented Media
2
Quantifying ECM Components in Tissue Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell free scaffolds (Day 0) and FPSC seeded constructs (Day 28) were analysed for DNA and sGAG content. Prior to performing assays, scaffolds and constructs were enzymatically digested with papain (125µg/ml -Sigma) in a buffer containing 100mM Sodium Phosphate (Sigma) with 5mM Na2EDTA (Sigma) at pH 6.5 as previously described 43 . sGAG quantification was performed using a 1, 9 dimethylmethylene blue (DMMB) assay according to the manufacturer's protocol with bovine tracheal chondroitin 4-sulfate used as a reference standard (Blyscan sulfated sGAG assay kit, Biocolor, Northern Ireland). Quantification of dsDNA in the digested constructs was performed using a Quant-iT Pico Green dsDNA kit (Invitrogen) according to the manufacturer's protocol. The combination of results from the DMMB and pico-green assays provides a ratio of sGAG normalized to dsDNA content.
Collagen content of scaffolds was quantified by measuring total hydroxyproline content as previously described 9 . A hydroxyproline:collagen ratio of 1:7.69 was assumed to determine the collagen content 50 .
Collagen content of scaffolds was quantified by measuring total hydroxyproline content as previously described 9 . A hydroxyproline:collagen ratio of 1:7.69 was assumed to determine the collagen content 50 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!