trap alp stain kit  (TaKaRa)

Journal: Clinical Implant Dentistry and Related Research

Article Title: Bone Morphogenetic Protein ( BMP ) 9 Outperforms BMP2 in Osteogenesis and Osseointegration: In Vitro and In Vivo

doi: 10.1111/cid.70135

Figure Lengend Snippet: Comparative effects of BMP9 and BMP2 on osteogenic differentiation and osteoclastogenesis in vitro. (A) Real‐time PCR analysis of key osteogenic genes (Col1, Runx2, ALP, and OCN) in MC3T3‐E1 cells treated with 8 nM of BMP2 or BMP9 for 3, 5, and 7 days. All gene‐expression levels were normalized to GAPDH. (B) Western blot analysis of osteogenic marker proteins in cell lysates harvested after 7 days of treatment with BMP2 or BMP9. GAPDH was used as the loading control. Densitometric quantification of band intensities (integrated density) normalized to GAPDH is shown below the blots and presented as relative protein expression. (C) Western blot showing dose‐dependent p‐Smad1/5/9 in MC3T3‐E1 cells exposed to varying concentrations of BMP2 or BMP9. Phosphorylation was quantified by densitometry and expressed as fold change vs. control after normalization using [(p‐Smad1/5/9)/(total Smad1/5/9)] and further normalized to GAPDH, as shown in the graph below the blots. Asterisks indicate statistical significance for pairwise comparisons between BMP2 and BMP9 at the same concentration (****, p < 0.0001), unless otherwise indicated. (D) ALP activity and representative images of ALP staining in MC3T3‐E1 cultures after 7 days of induction with BMP2 or BMP9. (E) Alizarin Red S staining illustrating mineralized nodule formation after extended culture with BMP2 or BMP9. (F) Representative TRAP‐stained images of RAW 264.7‐derived osteoclasts following treatment with RANKL (3 nM), BMP2 (8 nM), or BMP9 (8 nM) for 5 days. TRAP‐positive multinucleated osteoclasts are indicated by arrows. Scale bar, 20 μm. (G) Quantification of TRAP‐positive multinucleated cells per well. Data are presented as the mean ± SD ( n = 3 independent experiments), and p ‐values were calculated using one‐way analysis of variance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). BMP, bone morphogenetic protein; PCR, polymerase chain reaction; ALP, alkaline phosphatase; Col1, collagen type I; Runx2, runt‐related transcription factor 2; OCN, osteocalcin; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

Article Snippet: Recombinant human RANKL (11682‐HNCH; Sino Biological, Beijing, China) and a tartrate‐resistant acid phosphatase (TRAP) staining kit (MK300; Takara Bio, Shiga, Japan) were used for the osteoclast differentiation assay.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Marker, Control, Expressing, Phospho-proteomics, Concentration Assay, Activity Assay, Staining, Derivative Assay, Polymerase Chain Reaction

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) inhibits receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. a), c), and e) Immunofluorescence staining confirmed that CXCL5 (1 or 50 ng/ml) inhibits RANKL-induced differentiation of RAW264.7 cells into cathepsin K (CTSK)-positive osteoclast-like cells. b), d), and f) CXCL5 decreased the differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells. Purple indicates TRAP expression, and cyan indicates nuclei. g) and h) Western blot analysis showed that CXCL5 (1 ng/ml) inhibited RANKL-induced differentiation of RAW264.7 cells by suppressing the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), resulting in decreased CTSK expression at 72 hours of treatment. Data are presented as the mean (standard error of the mean (SEM)) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Osteoclast formation was evaluated by immunostaining for cathepsin K (CTSK) (ab19027; Abcam, UK) and tartrate-resistant acid phosphatase (TRAP) (MK300; Takara Bio, Japan).

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Comparison