Cell Proliferation, GAG, and ALP Assays

Biochemical assays were used to investigate cell proliferation, formation of glycosaminoglycans (GAG) and alkaline phosphatase (ALP) activity at 3, 7, 14 and 21 d as described earlier and following the respective user's manual [44 (link)]. Adenosine triphosphate (ATP) assays using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) were used to examine cell proliferation. After digestion with papain (1 μg/ml; Sigma) the content of GAG in harvested pellets was measured with the Blyscan™ Sulfated Glycosaminoglycan Assay (Biocolor Ltd, Newtownabbey, Northern Ireland) by reaction with 1,9-dimethylmethylene blue. The ALP activity was measured in an Enzyme-linked Immunosorbent Assay (ELISA) reader by the conversion of p-nitrophenol-phosphate to p-nitrophenol and inorganic phosphate via absorbance at 405 nm to determine the Quant-iT™ PicoGreen® kit (Invitrogen GmbH, Darmstadt, Germany).

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Weißenberger M., Wagenbrenner M., Nickel J., Ahlbrecht R., Blunk T., Steinert A.F, & Gilbert F. (2023). Comparative in vitro treatment of mesenchymal stromal cells with GDF-5 and R57A induces chondrogenic differentiation while limiting chondrogenic hypertrophy. Journal of Experimental Orthopaedics, 10, 29.

Publication 2023

CellTiter-Glo® Luminescent Cell Viability Assay papain Blyscan™ Sulfated Glycosaminoglycan Assay Quant-iT™ PicoGreen® kit

Adenosine triphosphate Alkaline phosphatase Assay Cell proliferation Cell viability Digestion Dimethylmethylene blue Enzyme linked immunosorbent assay Glycosaminoglycans Inorganic phosphate Luminescent assay Nitrophenol P nitrophenol Papain Pellets Picogreen Promega Sulfated glycosaminoglycan

Corresponding Organization : Klinik König-Ludwig Haus

Other organizations : Ludwig-Maximilians-Universität München, Universitätsklinikum Würzburg

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Variable analysis

independent variables

Time (3, 7, 14, 21 days)

dependent variables

Cell proliferation
Glycosaminoglycans (GAG) formation
Alkaline phosphatase (ALP) activity

control variables

Biochemical assays performed as described earlier and following the respective user's manual [44 (link)]

controls

Positive control: Not specified
Negative control: Not specified

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