MCB-22-174

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

3T3-L1 preadipocytes are induced to differentiate after growth arrest using adipogenic media containing insulin, dexamethasone, and IBMX; differentiation is assessed by lipid-droplet accumulation, triglyceride increase, Oil Red O staining, and adipocyte-marker induction such as PPARγ and C/EBPα.

MSC chondrogenic differentiation is commonly induced by culturing bone marrow-derived mesenchymal stromal/stem cells as high-density three-dimensional pellets or micromass aggregates in defined chondrogenic medium containing TGF-β family stimulation; the readout is formation of cartilage-like extracellular matrix, especially sulfated proteoglycans, aggrecan, and type II collagen. The assay detects chondrogenesis by pellet enlargement, metachromatic or Alcian blue/Safranin O staining of proteoglycan-rich matrix, immunodetection of type II collagen and aggrecan, and gene-expression changes in cartilage matrix markers; hypertrophic or fibrocartilaginous drift can be assessed by collagen X and collagen I readouts when included.

Ca²⁺ staining is an experimental technique that utilizes specific fluorescent probes (such as Fluo-4 AM, Fura-2, etc.) to qualitatively or quantitatively detect dynamic changes in intracellular Ca²⁺ concentrations; this is achieved by monitoring the changes in fluorescent signals generated when these probes bind to free intracellular calcium ions. The underlying principle relies primarily on the presence of chelating groups within the probe's molecular structure that possess high affinity for calcium ions.

Lipid droplets are intracellular organelles with a neutral-lipid core that stores triacylglycerols and sterol esters, and Oil Red O or Sudan dyes detect these hydrophobic lipid deposits by partitioning into retained lipids in fresh or frozen specimens. Oil Red O stains neutral triglycerides and lipids in frozen tissue sections or air-dried cytologic preparations, while Sudan Black B has also been used as a histochemical fat stain for lipid-rich tissue structures.