The cells were subjected to induced differentiation using commercial kits (StemPro A10070-01, A10071-01, and A10072-01; Gibco) according to the manufacturer's instructions. Briefly, for the chondrogenic cell differentiation, the eBMmsc, eADmsc, and eUCmsc were plated in 5 μl droplets at a concentration of 1.6 × 107 cells/ml and cultured under standard conditions for 2 h; then, the differentiation medium was added. The medium was replaced every 4 days for 2 weeks. For the osteogenic differentiation, 5 × 103 cells/cm2 were plated and incubated for 24 h; then, the IMDM medium was replaced by a specific differentiation medium, which was replaced every 4 days for 2 weeks. For the adipogenic differentiation, 1 × 104 cells/cm2 were plated and incubated for 24 h; then, the IMDM medium was replaced by a specific differentiation medium, which was replaced every four days for two weeks. The cells in the control group were plated under the same conditions but were maintained in IMDM medium. At the end of the two-week period, the cells were fixed and stained using Sudan black dye (Sigma-Aldrich) for the adipogenesis differentiation detection, Alizarin red (Sigma-Aldrich) for the osteogenesis detection, and Alcian blue (Sigma-Aldrich) for the chondrogenesis differentiation detection.
Sudan black dye
Manufactured by Merck Group
Sourced in United States
Sudan black dye is a fat-soluble dye commonly used in histological and cytological staining procedures. It is primarily employed to visualize lipids, especially neutral lipids and phospholipids, within biological samples. The dye exhibits a deep blue-black color and can be applied to a variety of specimen types, including tissues, cells, and prepared slides.
Automatically generated - may contain errors
2 protocols using sudan black dye
1
Differentiating Mesenchymal Stem Cells
2
Lipofuscin Staining in Aged Testes
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Lipofuscin staining was performed using the Sudan black dye (Sigma, Saint Louis, MO, USA) on a subset of the testes histological slides from each mouse. Lipofuscin is a heterogeneous pigmented by-product due to failure of intracellular catabolism, conventionally found in the lysosomes or cytosol of post-mitotic aged cells [25] . We used a modified protocol [26] (link). Briefly, the histological slides were dewaxed with xylol, washed in an alcohol gradient until reaching 70% alcohol and rehydrated in water. After diluting the Sudan black in 70% alcohol, avoiding its precipitation, a 10 mL syringe with a filter was used to drop the Sudan black solution on a clean slide, which was positioned downwards over the slide with the tissue for approximately 2 min. The slide was then washed with 50% alcohol and distilled water. A glycerol droplet and a coverslip were placed over the section and observed under the light microscope. Images obtained at 10X magnification (3 sections per mouse) were used to quantify the lipofuscin positive area (dark blue). The area of pixels in the images was calculated using the Image J software (National Institutes of Health -USA) and presented as percentage relative to the total area of the section.
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