anti cd51 61 antibody  (Santa Cruz Biotechnology)

Journal: Advanced Science

Article Title: Osteoclast‐Derived SLIT3 Mediates Osteoarthritis Pain and Degenerative Changes

doi: 10.1002/advs.202517545

Figure Lengend Snippet: SLIT3 is mainly derived from osteoclasts. a) Representative images of SLIT3 (green) and CD51/61 (red) co‐stained cells in subchondral bone in the CON and UAC groups. The white dashed line represents the boundary between bone and cartilage. Scale bars: 10 µm. b) Quantitative analysis in panel (a). n = 6. c–e) FACS of the markers of BMSCs (c), osteoblasts (d), osteoclasts (e) and BMMs. f) from the mandible in 6‐week‐old female UAC rats. n = 6. g) The average Ct value and h) qRT‐PCR analysis of the expression of the Slit3 in BMSCs, osteoblasts, osteoclasts, and BMMs. n = 3. Statistical analyses were performed using Student's t ‐test. **** p < 0.0001. ns : no significance.

Article Snippet: The antibodies used were Protein gene product 9.5 (PGP9.5, 1:400, ab8189, Abcam, Cambridge, UK), calcitonin gene‐related peptide 1 (CGRP, 1:300, sc‐57053, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SLIT3 (1:300, DF9909, Affinity Biosciences, Cincinnati, OH, USA), TRAP‐α (1:300, sc‐373916, Santa Cruz Biotechnology), Osteocalcin (1:300, sc‐74495, Santa Cruz Biotechnology), β3‐tubulin (1:300, Cell Signaling Technology, Danvers, MA, USA) and CD51/61(1:300, AF5152, Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Derivative Assay, Staining, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: Osteoclast‐Derived SLIT3 Mediates Osteoarthritis Pain and Degenerative Changes

doi: 10.1002/advs.202517545

Figure Lengend Snippet: Slit3 is specifically knocked out in osteoclasts of Slit3 ‐CKO mice. a) Schematic diagram of Slit3 ‐CKO mice. b) Immunofluorescence of SLIT3 (green) and CD51/61 (red) in subchondral bone. The white dashed line represents the boundary between subchondral bone and cartilage. Scale bars:10 µm. c) Quantitative analysis in panel (b). n = 6. d) qRT‐PCR analysis of the gene expression of Slit3 in subchondral bone in the two groups. n = 3. e,f) Western blot and its quantitative analysis, g) ELISA, and h,i) immunohistochemical staining and quantitative analysis of SLIT3 expression in subchondral bone in the WT and Slit3 ‐CKO groups. n = 3. Scale bars: 70 µm. Statistical analyses were performed using Student's t ‐test. * p < 0.05. ** p < 0.01. *** p < 0.001. **** p <0.0001. ns : no significance.

Article Snippet: The antibodies used were Protein gene product 9.5 (PGP9.5, 1:400, ab8189, Abcam, Cambridge, UK), calcitonin gene‐related peptide 1 (CGRP, 1:300, sc‐57053, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SLIT3 (1:300, DF9909, Affinity Biosciences, Cincinnati, OH, USA), TRAP‐α (1:300, sc‐373916, Santa Cruz Biotechnology), Osteocalcin (1:300, sc‐74495, Santa Cruz Biotechnology), β3‐tubulin (1:300, Cell Signaling Technology, Danvers, MA, USA) and CD51/61(1:300, AF5152, Affinity Biosciences, Cincinnati, OH, USA).

Techniques: Immunofluorescence, Quantitative RT-PCR, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Oncology

Article Title: Spontaneous Osteoclastogenesis, a risk factor for bone metastasis in advanced luminal A-type breast cancer patients

doi: 10.3389/fonc.2023.1073793

Figure Lengend Snippet: Bone marrow (BM) and peripheral blood (PB) spontaneously derived osteoclasts (OCs) are mature and functional. (A) Representative pictures showing cell surface antigens MMP-9, c-Src and αV integrin (CD51) identifying mature OC cultured from a breast cancer patient (BCP) on bovine cortical bone slices by immunocytochemistry. Scale bars: 100 µm. (B) Arrows show resorption lacunae acidification (acid vacuoles) on BCP derived OC by Acridine Orange staining. Acridine Orange is green at neutral pH and red at acidic pH. Scale bars: 100 µm. (C) Representative pictures showing BCP-BM derived OC resoption lacunae formation on bovine cortical slices. After OC removal, cortical bone slices were stained with Toluidine Blue; arrows indicate resoption lacunae. Scale bars: 100 µm.

Article Snippet: Single labelling using primary Abs anti-human: anti-RANK (RANKL receptor, mouse IgG1, MAB683, R&D Systems), anti-CD11b-PE (mouse IgG2ak, 347557, BD Bioscience), anti-CD14 (mouse IgG1k, 14-0149, eBioscience), anti- CD51/61-FITC (integrin alphaV/beta3, mouse IgG1k, 555505, BD Bioscience) and anti-CD115 (or c-fms, M-CSF receptor, rat IgG1, 4-1159, eBioscience) were performed.

Techniques: Derivative Assay, Functional Assay, Cell Culture, Immunocytochemistry, Staining

Journal: Frontiers in Oncology

Article Title: Spontaneous Osteoclastogenesis, a risk factor for bone metastasis in advanced luminal A-type breast cancer patients

doi: 10.3389/fonc.2023.1073793

Figure Lengend Snippet: BM-MNCs and PB-MNCs phenotypic profile.

Article Snippet: Single labelling using primary Abs anti-human: anti-RANK (RANKL receptor, mouse IgG1, MAB683, R&D Systems), anti-CD11b-PE (mouse IgG2ak, 347557, BD Bioscience), anti-CD14 (mouse IgG1k, 14-0149, eBioscience), anti- CD51/61-FITC (integrin alphaV/beta3, mouse IgG1k, 555505, BD Bioscience) and anti-CD115 (or c-fms, M-CSF receptor, rat IgG1, 4-1159, eBioscience) were performed.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: IL-23 induces human osteoclastogenesis via IL-17 in vitro , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

doi: 10.1186/ar2297

Figure Lengend Snippet: Formation of human osteoclasts from PBMC induced by rhIL-23 (1.0 ng/ml) and M-CSF. As negative and positive controls, peripheral blood mononuclear cells (PBMC) were cultured with macrophage-colony stimulating factor (M-CSF) alone during the first 3 days; as a negative control, adherent cells were then cultured with only M-CSF (a) , and as a positive control they were cultured with M-CSF plus soluble receptor activator of NF-κB ligand (sRANKL; 100 ng/ml) (b) for the last 7 days. Human osteoclasts induced from PBMC by recombinant human (rh)IL-23 (1.0 ng/ml) and M-CSF were detected by staining with tartrate-resistant acid phosphatase (TRAP) (c) and immunohistological staining by vitronectin receptor αvβ3 (CD51/61) (d) . (e) Osteoclasts induced from PBMC by rhIL-23 (1.0 ng/ml) were also evaluated functionally by pit formation on Osteologic ® . Original magnification ×100.

Article Snippet: Anti-human CD51/61 mAb was purchased from BD Bioscience Pharmingen (San Diego, CA, USA).

Techniques: Cell Culture, Negative Control, Positive Control, Recombinant, Staining

Journal: Arthritis Research & Therapy

Article Title: IL-23 induces human osteoclastogenesis via IL-17 in vitro , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

doi: 10.1186/ar2297

Figure Lengend Snippet: Inhibition of IL-23-induced osteoclastogenesis by osteoprotegerin, anti-IL-17 antibody, and etanercept. Peripheral blood mononuclear cells (PBMC) were cultured during the first 3 days with macrophage-colony stimulating factor (M-CSF) and recombinant human (rh)IL-23 (1.0 ng/ml) (c) . At the same time, osteoprotegerin (OPG, 250 ng/ml) (d) , anti-IL-17 antibody (5 μg/ml) (e) , or etanercept (0.01 μg/ml) (f) was added with rhIL-23 (1.0 ng/ml). Adherent cells were cultured with M-CSF alone during the last 7 days (days 4 to 10 (c–f) ). As controls, PBMC were cultured with M-CSF alone during the first 3 days, after which adherent cells were cultured with M-CSF only (a) (negative control) or with soluble receptor activator of NF-κB ligand (sRANKL; 100 ng/ml) (b) (positive control). Osteoclasts were detected by immunohistological staining for vitronectin receptor αvβ3 (CD51/61). Original magnification ×100.

Article Snippet: Anti-human CD51/61 mAb was purchased from BD Bioscience Pharmingen (San Diego, CA, USA).

Techniques: Inhibition, Cell Culture, Recombinant, Negative Control, Positive Control, Staining

Journal: Cells

Article Title: A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups

doi: 10.3390/cells11243973

Figure Lengend Snippet: Human osteoclasts differentiated in 3D form a more homogeneous cell population than monolayer cells. ( A – E ) Monolayer osteoclasts : ( A ) Representative images of staining for CD51/61 (top) and visualisation of resorption tracks (bottom, scale bars = 200 µm) and ( B ) TRAP staining of osteoclasts from 4 independent donors (scale bar = 200 µm) on day 9 of differentiation showing heterogeneity of the populations; (insert) monocytes (red arrow), binuclear pre-osteoclasts (blue arrow) and mature osteoclasts (green arrow). ( C ) Rate of formation of monolayer osteoclasts (TRAP-positive cells with ≥3 nuclei) and ( D ) the size distribution of these osteoclasts; small (3–5 nuclei), medium (6–9 nuclei) or large (>10 nuclei). ( E ) Representative TRAP-stained images of the time course of osteoclast differentiation in monolayer culture (scale bar = 100 µm). ( F – H ) 3D-generated osteoclasts: Cells were fixed for analysis 4 h after reseeding onto cell culture plates ( F ) Rate of formation of 3D-generated osteoclasts (TRAP-positive cells with ≥3 nuclei) and ( G ) the size distribution of these osteoclasts; small (3–5 nuclei), medium (6–9 nuclei) or large (>10 nuclei). ( H ) Representative TRAP-stained images of the time course of osteoclast differentiation in 3D (scale bar = 100 µm). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: CD51/61 staining: immunostaining for osteoclast-specific CD51/61 used an anti-CD51/61 antibody (clone 23C6, 1:400; BioRad, Oxford, UK) and standard DAB immunohistochemistry techniques.

Techniques: Staining, Generated, Cell Culture

Journal: Cells

Article Title: A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups

doi: 10.3390/cells11243973

Figure Lengend Snippet: Cell surface markers in monolayer and 3D osteoclasts. ( A ) FACS scatter graphs showing increasing cell size and complexity during differentiation from CD14+ monocytes to osteoclasts in 3D culture. ( B ) Representative data from cells from one donor showing variation in osteoclast surface markers over 24 days of differentiation. Black = monolayer cells, red = 3D-generated cells. ( C , D ) Comparison of expression of ( C ) CD9 (n = 3) and ( D ) CD51/61 (n = 5) during 3D differentiation of osteoclasts from different monocyte donors. ( E ) Double staining of 3D-generated osteoclasts on day 15 of differentiation for CD9 and CD51/61 distinguishes two osteoclast subpopulations. ( F ) Scatter graph of CD9+CD51/61−cells showing small early/pre-osteoclasts (left panel) with CD9+CD51/61+ cells being larger mature osteoclasts (right panel). ***, p < 0.001.

Article Snippet: CD51/61 staining: immunostaining for osteoclast-specific CD51/61 used an anti-CD51/61 antibody (clone 23C6, 1:400; BioRad, Oxford, UK) and standard DAB immunohistochemistry techniques.

Techniques: Generated, Expressing, Double Staining

Journal: Cells

Article Title: A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups

doi: 10.3390/cells11243973

Figure Lengend Snippet: Live cell sorting of primary human osteoclasts. ( A ) Flow cytometry of live 3D-generated human osteoclasts stained for CD51/61. ( B ) Following cell sorting, the population of CD51/61+ cells increased from 13.3% to 84.2%. ( C ) Cells re-seeded into monolayer culture after live cell sorting and analysed for the presence of osteoclasts (TRAP stain, 24 h post re-seed, scale bar = 100 µm) and resorption activity (resorption tracks on dentine, 7 days post re-seed, scale bar = 200 µm). Top panel: CD51/61-positive cells from 3D culture. Middle panel: CD51/61-negative cells from 3D culture. Bottom panel: CD51/61-positive cells from monolayer culture. ( D ) Flow cytometry of live 3D-generated human osteoclasts stained for CD9 and CD51/61. CD9+CD51/61- and CD9+CD51/61+ cells were re-seeded into monolayer culture after live cell sorting and analysed for the presence of osteoclasts 24 h post re-seed (TRAP stain, scale bar = 100 µm), quantified ( E ) as the number of nuclei per cell in these selected populations. ( F ) Area of bone resorbed by monolayer and 3D-generated osteoclasts selected for CD51/61. Black = monolayer cells, red = 3D-generated cells. ***, p < 0.001.

Article Snippet: CD51/61 staining: immunostaining for osteoclast-specific CD51/61 used an anti-CD51/61 antibody (clone 23C6, 1:400; BioRad, Oxford, UK) and standard DAB immunohistochemistry techniques.

Techniques: FACS, Flow Cytometry, Generated, Staining, Activity Assay