Alcian blue solution 8x

Early (P3-15) passage cells were seeded in standard MSC medium in 24-well plates (1.25x10⁴ cells/well) and grown to 90% confluence. The medium was subsequently replenished with either control (MesenCult-ACF Chondrogenic Differentiation Basal Medium (STEMCELL Technologies, Vancouver, Canada), 2mM L-glutamine) or differentiation (MesenCult-ACF Chondrogenic Differentiation Basal Medium, 2mM L-glutamine, MesenCult-ACF 20X Chondrogenic Differentiation Supplement) medium and incubated at 37°C/5% CO₂ for 18 days. On day 18, the cells were fixed in 10% neutral buffered formalin and stained with Alcian blue solution (8x, pH2.5) (Sigma-Aldrich, Sydney, Australia). Chondrogenesis was visualised by Alcian blue staining of filamentous glycosaminoglycans.

Early, mid, and late passage cells were seeded in 24-well plates (1.25 × 10⁴ cells/well) and grown to 90% confluence in standard MSC medium. The medium was subsequently replenished with either control (MesenCult™-ACF Chondrogenic Differentiation Basal Medium (STEMCELL Technologies^®, Vancouver, Canada) with 2 mM L-glutamine) or differentiation (MesenCult™-ACF Chondrogenic Differentiation Basal Medium, 2 mM L-glutamine, MesenCult™-ACF 20X Chondrogenic Differentiation Supplement) medium and incubated at 37°C/5% CO₂ for 18 days. On day 18, the cells were fixed in 10% v/v neutral buffered formalin and stained with Alcian blue solution (8x, pH 2.5) (Sigma-Aldrich™, Sydney, Australia). Chondrogenesis was visualised by Alcian blue staining of filamentous glycosaminoglycans.