trap staining commercial kit  (Beijing Solarbio Science)

Journal: Journal of Microbiology and Biotechnology

Article Title: Inhibitory Effect of Biotransformed-Fucoidan on the Differentiation of Osteoclasts Induced by Receptor for Activation of Nuclear Factor-κB Ligand

doi: 10.4014/jmb.2203.03001

Figure Lengend Snippet: ( A ) Cell viabilities of osteoclasts; ( B ) TRAP activities of osteoclasts; ( C ) Detection of TRAP-positive multinucleated cells. Bone marrow-derived macrophages (BMMs) were cultured for 2 days in the presence of M-CSF and then further treated with M-CSF and RANKL for 3 days to differentiate osteoclasts. Cytotoxicities were determined using an MTT assay. To measure TRAP activity, differentiated osteoclasts were cultured with 2 μg/ml of three ultrafiltrated Fu/Pec fractions in the presence of RANKL for 3 days. Cells were then treated with TRAP substrate solution and TRAP activities were determined by measuring optical densities at 405 nm. To observe TRAP-positive multinucleated cells with more than 3 of the number of nuclei, differentiated osteoclasts prepared with same method for TRAP activity were stained using a commercial TRAP staining kit. Cells were photographed under a Leica DM inverted microscope system (Wetzlar, Germany). The data represented the mean value±SEM of analyses performed in triplicate. *** p < 0.001 vs. RANKL only and non-treated group. Fu/Pec: fucoidan hydrolyzed with Pectinex.

Article Snippet: In addition, to visualize TRAP activity through visualized images, we used a commercial TRAP staining kit (Sigma Chemicals) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, MTT Assay, Activity Assay, Staining, Inverted Microscopy

Journal: Scientific Reports

Article Title: D-galactose-induced aging aggravates obesity-induced bone dyshomeostasis

doi: 10.1038/s41598-022-12206-4

Figure Lengend Snippet: D-galactose-induced senescence and hyperinsulinism-stimulated insulin resistance accelerated RANKL-induced osteoclast differentiation in RAW 264.7 cells. ( a ) % cell viability of RAW 264.7 belonging to four treatment group, ( b ) Number of TRAP-positive multinucleated cells which presented more than three nuclei indicated a mature osteoclast. ( c ) TRAP positive staining of RANKL-stimulated multinucleated osteoclasts with the treatments of control, 5 mg/mL D-galactose, 100 nM insulin and the combined treatment (20 × magnification). Bar graphs presented as mean ± SEM (n = 6/group).* p < 0.05 versus control group. D-gal, D-galactose; RANKL, receptor activator of nuclear factor κB ligand; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: A commercial TRAP staining kit (Cosmo Bio, Tokyo, Japan) was used at 37 °C for 60 min to determine osteoclast formation.

Techniques: Staining