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Immunodiagnostic Systems mouse trap (trap 5b) elisa kit
Mouse Trap (Trap 5b) Elisa Kit, supplied by Immunodiagnostic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+trap+assay+kit/mouse+free+soluble++s+rankl+elisa+assay+kit/pmc11235167-249-13-21
Average 90 stars, based on 1 article reviews
mouse trap (trap 5b) elisa kit - by Bioz Stars, 2026-10
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Related Articles

ALP Assay:

Article Title: Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma
Article Snippet: .. The serum levels of calcium and alkaline phosphatase (ALP) were measured using a QuantiChrome Calcium and ALP assay kit (BioAssay Systems, Hayward, CA, USA) and the serum level of tartrate-resistant acid phosphatase (TRAP) 5b was determined with a mouse TRAP assay kit (Immunodiagnostic Systems, Boldon, UK) according to the manufacturer's instructions. ..

TRAP Assay:

Article Title: Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma
Article Snippet: .. The serum levels of calcium and alkaline phosphatase (ALP) were measured using a QuantiChrome Calcium and ALP assay kit (BioAssay Systems, Hayward, CA, USA) and the serum level of tartrate-resistant acid phosphatase (TRAP) 5b was determined with a mouse TRAP assay kit (Immunodiagnostic Systems, Boldon, UK) according to the manufacturer's instructions. ..

Article Title: Treatment of bone diseases
Article Snippet: Histomorphometry was done with a semi-automatic image analysing system combining a microscope equipped with a camera lucida and digitizing tablet linked to a computer using the OsteoMeasure Software (Osteometrics Inc., Decatur, Ga., USA). .. TRAP was measured in 20 μl of serum to assess osteoclastic activity using the Mouse TRAP Assay kit (Immunodiagnostic Systems Ltd., Scottsdale, Ariz.). (vii) Western Blot Analysis Marrow cells were obtained from the left femur of PA- and vehicle-treated animals (n=6 per group) by flushing with Dulbecco's alpha modified medium (DMEM). .. 100 mg of tissue were dissolved in nitric acid and concentrations of nine minerals was quantified in the tissue homogenates using an Agilent 5973 mass selective detector via an auto-sampler Agilent Technologies 7683, and controlled using Agilent ChemStation software (Agilent, Santa Clara, Calif., USA).

Article Title: VDR in Osteoblast-Lineage Cells Primarily Mediates Vitamin D Treatment-Induced Increase in Bone Mass by Suppressing Bone Resorption.
Article Snippet: Serum FGF23 concentrations were quantified by a FGF23 ELISA kit (KAINOS laboratories, Tokyo, Japan). .. Serum tartrate-resistant acid phosphatase 5b (TRAP5b) activities were measured using a mouse TRAP assay kit (Immunodiagnostic Systems, Boldon, UK). ..

Article Title: Def6 restrains osteoclastogenesis and inflammatory bone resorption 1
Article Snippet: .. When the mice were sacrificed, serum was collected to determine the TRAP concentration using a Mouse TRAP Assay Kit (immunodiagnostic systems) according to the manufacturer’s instructions. ..

Article Title: Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts.
Article Snippet: .. Serum levels of calcium were determined using the QuantiChrome Calcium assay kit (BioAssay Systems, Hayward, CA), and those of TRAP 5b and C-terminal cross-linking telopeptide of type Ι collagen (CTX) were measured by mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK) and RatLaps enzyme immunoassay kit (Immuno Diagnostic Systems), respectively. .. Cell lines and cell cultureMDA-MB-231 human metastatic breast cancer and A549 human lung cancer cells (Korean cell line bank, Seoul, Korea) were grown in DMEM and RPMI 1640 medium, respectively, containing 10% FBS and 1% antibiotic-antimycotic mixture at 37°C with 5% CO2.

Article Title: Early estrogen‐induced gene 1 facilitates osteoclast formation through the inhibition of interferon regulatory factor 8 expression
Article Snippet: .. Tartrate-resistant acid phosphatase (TRAP) levels were determined using a Mouse TRAP Assay kit (Immunodiagnostic Systems, Tyne and Wear, UK) in accordance with the manufacturer's protocol. .. For histological analysis, the calvaria were fixed in formaldehyde (SigmaAldrich Corporation) for 24 hours and then decalcified in 0.5 M ethylenediaminetetraacetic acid (pH 7.4) (EDTA; LPS solution) at 4°C for 7 days.

Article Title: Human antigen R-regulated CCL20 contributes to osteolytic breast cancer bone metastasis
Article Snippet: .. The serum levels of TRAP 5b and CTX were measured with a mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK) and a RatLaps EIA kit (Immuno Diagnostic Systems) according to the manufacturer’s instructions. .. Cell viability, migration, and invasion were measured with MTT assay, wound-healing assay, and a transwell chamber (Corning Costar, Lowell, MA), respectively, as described in Supplementary materials and methods.

Article Title: Does a Treadmill Running Exercise Contribute to Preventing Deterioration of Bone Mineral Density and Bone Quality of the Femur in KK-Ay Mice, a Type 2 Diabetic Animal Model?
Article Snippet: Although it has been recently shown that type 2 diabetics have an increased risk of hip fracture, the effects of exercise therapy to prevent this have not been clarified.. We examined whether a treadmill running exercise contributes to the bone mineral density (BMD) and bone microarchitecture of the femur and what kind of exercise intensity and duration are optimum in type 2 diabetes mellitus using KK-Ay diabetic mice.. The mice were divided into two running groups, one fast speed and short duration (FS), the other slow speed and long duration (SL), and a group of controls with no running (CO).

Activity Assay:

Article Title: Treatment of bone diseases
Article Snippet: Histomorphometry was done with a semi-automatic image analysing system combining a microscope equipped with a camera lucida and digitizing tablet linked to a computer using the OsteoMeasure Software (Osteometrics Inc., Decatur, Ga., USA). .. TRAP was measured in 20 μl of serum to assess osteoclastic activity using the Mouse TRAP Assay kit (Immunodiagnostic Systems Ltd., Scottsdale, Ariz.). (vii) Western Blot Analysis Marrow cells were obtained from the left femur of PA- and vehicle-treated animals (n=6 per group) by flushing with Dulbecco's alpha modified medium (DMEM). .. 100 mg of tissue were dissolved in nitric acid and concentrations of nine minerals was quantified in the tissue homogenates using an Agilent 5973 mass selective detector via an auto-sampler Agilent Technologies 7683, and controlled using Agilent ChemStation software (Agilent, Santa Clara, Calif., USA).

Article Title: Does a Treadmill Running Exercise Contribute to Preventing Deterioration of Bone Mineral Density and Bone Quality of the Femur in KK-Ay Mice, a Type 2 Diabetic Animal Model?
Article Snippet: Although it has been recently shown that type 2 diabetics have an increased risk of hip fracture, the effects of exercise therapy to prevent this have not been clarified.. We examined whether a treadmill running exercise contributes to the bone mineral density (BMD) and bone microarchitecture of the femur and what kind of exercise intensity and duration are optimum in type 2 diabetes mellitus using KK-Ay diabetic mice.. The mice were divided into two running groups, one fast speed and short duration (FS), the other slow speed and long duration (SL), and a group of controls with no running (CO).

Western Blot:

Article Title: Treatment of bone diseases
Article Snippet: Histomorphometry was done with a semi-automatic image analysing system combining a microscope equipped with a camera lucida and digitizing tablet linked to a computer using the OsteoMeasure Software (Osteometrics Inc., Decatur, Ga., USA). .. TRAP was measured in 20 μl of serum to assess osteoclastic activity using the Mouse TRAP Assay kit (Immunodiagnostic Systems Ltd., Scottsdale, Ariz.). (vii) Western Blot Analysis Marrow cells were obtained from the left femur of PA- and vehicle-treated animals (n=6 per group) by flushing with Dulbecco's alpha modified medium (DMEM). .. 100 mg of tissue were dissolved in nitric acid and concentrations of nine minerals was quantified in the tissue homogenates using an Agilent 5973 mass selective detector via an auto-sampler Agilent Technologies 7683, and controlled using Agilent ChemStation software (Agilent, Santa Clara, Calif., USA).

Modification:

Article Title: Treatment of bone diseases
Article Snippet: Histomorphometry was done with a semi-automatic image analysing system combining a microscope equipped with a camera lucida and digitizing tablet linked to a computer using the OsteoMeasure Software (Osteometrics Inc., Decatur, Ga., USA). .. TRAP was measured in 20 μl of serum to assess osteoclastic activity using the Mouse TRAP Assay kit (Immunodiagnostic Systems Ltd., Scottsdale, Ariz.). (vii) Western Blot Analysis Marrow cells were obtained from the left femur of PA- and vehicle-treated animals (n=6 per group) by flushing with Dulbecco's alpha modified medium (DMEM). .. 100 mg of tissue were dissolved in nitric acid and concentrations of nine minerals was quantified in the tissue homogenates using an Agilent 5973 mass selective detector via an auto-sampler Agilent Technologies 7683, and controlled using Agilent ChemStation software (Agilent, Santa Clara, Calif., USA).

Concentration Assay:

Article Title: Def6 restrains osteoclastogenesis and inflammatory bone resorption 1
Article Snippet: .. When the mice were sacrificed, serum was collected to determine the TRAP concentration using a Mouse TRAP Assay Kit (immunodiagnostic systems) according to the manufacturer’s instructions. ..

Quantichrome Calcium Assay:

Article Title: Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts.
Article Snippet: .. Serum levels of calcium were determined using the QuantiChrome Calcium assay kit (BioAssay Systems, Hayward, CA), and those of TRAP 5b and C-terminal cross-linking telopeptide of type Ι collagen (CTX) were measured by mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK) and RatLaps enzyme immunoassay kit (Immuno Diagnostic Systems), respectively. .. Cell lines and cell cultureMDA-MB-231 human metastatic breast cancer and A549 human lung cancer cells (Korean cell line bank, Seoul, Korea) were grown in DMEM and RPMI 1640 medium, respectively, containing 10% FBS and 1% antibiotic-antimycotic mixture at 37°C with 5% CO2.

Enzyme-linked Immunosorbent Assay:

Article Title: Artemisinin-Daumone Hybrid Inhibits Cancer Cell-Mediated Osteolysis by Targeting Cancer Cells and Osteoclasts.
Article Snippet: .. Serum levels of calcium were determined using the QuantiChrome Calcium assay kit (BioAssay Systems, Hayward, CA), and those of TRAP 5b and C-terminal cross-linking telopeptide of type Ι collagen (CTX) were measured by mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK) and RatLaps enzyme immunoassay kit (Immuno Diagnostic Systems), respectively. .. Cell lines and cell cultureMDA-MB-231 human metastatic breast cancer and A549 human lung cancer cells (Korean cell line bank, Seoul, Korea) were grown in DMEM and RPMI 1640 medium, respectively, containing 10% FBS and 1% antibiotic-antimycotic mixture at 37°C with 5% CO2.

Article Title: Human antigen R-regulated CCL20 contributes to osteolytic breast cancer bone metastasis
Article Snippet: .. The serum levels of TRAP 5b and CTX were measured with a mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK) and a RatLaps EIA kit (Immuno Diagnostic Systems) according to the manufacturer’s instructions. .. Cell viability, migration, and invasion were measured with MTT assay, wound-healing assay, and a transwell chamber (Corning Costar, Lowell, MA), respectively, as described in Supplementary materials and methods.

Article Title: Does a Treadmill Running Exercise Contribute to Preventing Deterioration of Bone Mineral Density and Bone Quality of the Femur in KK-Ay Mice, a Type 2 Diabetic Animal Model?
Article Snippet: Although it has been recently shown that type 2 diabetics have an increased risk of hip fracture, the effects of exercise therapy to prevent this have not been clarified.. We examined whether a treadmill running exercise contributes to the bone mineral density (BMD) and bone microarchitecture of the femur and what kind of exercise intensity and duration are optimum in type 2 diabetes mellitus using KK-Ay diabetic mice.. The mice were divided into two running groups, one fast speed and short duration (FS), the other slow speed and long duration (SL), and a group of controls with no running (CO).

Marker:

Article Title: Does a Treadmill Running Exercise Contribute to Preventing Deterioration of Bone Mineral Density and Bone Quality of the Femur in KK-Ay Mice, a Type 2 Diabetic Animal Model?
Article Snippet: Although it has been recently shown that type 2 diabetics have an increased risk of hip fracture, the effects of exercise therapy to prevent this have not been clarified.. We examined whether a treadmill running exercise contributes to the bone mineral density (BMD) and bone microarchitecture of the femur and what kind of exercise intensity and duration are optimum in type 2 diabetes mellitus using KK-Ay diabetic mice.. The mice were divided into two running groups, one fast speed and short duration (FS), the other slow speed and long duration (SL), and a group of controls with no running (CO).



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Fig. 1 Aged bone marrow-derived mesenchymal stem cells (BMMSCs) showed degenerative properties in osteogenesis and melatonin (MEL) promoted osteogenesis of aged BMMSCs. a) Haematoxylin and eosin (H&E) staining (40×) of proximal tibiae and b) micro-CT (μCT) imaging of distal femora of aged and young mice. c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). An independent-samples t-test was used to calculate the p-value in Figure 1c. d) Alkaline phosphatase (ALP) staining (20×) and e) Alizarin Red staining (20×) to analyze the osteogenesis of young and aged BMMSCs. f) Gene enrichment analysis of genes related to senile osteoporosis predicted using the Genecard database, DisGeNET database, and OMIM database, highlighting pathways where MEL metabolism and effects showed the highest enrichment in WikiPathways. Chi-squared test was used to calculate the p-value. g) MEL in serum was analyzed by enzyme-linked immunosorbent assay <t>(ELISA).</t> h) ALP staining (5×), i) ALP level analysis, j) Alizarin Red staining (5×), and k) mineralization level analysis were performed. *p < 0.05, significant differences between each indicated group analyzed using one-way analysis of variance (ANOVA). CTR, control group; IL-18, interleukin-18; MAPK, mitogen-activated protein kinase; ns, not significant; PI3K-AKT, phosphoinositide 3-kinase-protein kinase B; VEGFA-VEGFR2, vascular endothelial growth factor-vascular endothelial growth factor receptor 2.
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RAM2061 does not significantly impact normal bone remodeling in CD-1 mice. (A) Schematic representation of drug administration/dosing and downstream readouts conducted. Abbreviations: procollagen type 1 N-propeptide (P1NP); C-terminal telopeptide (CTX), tartrate resistant acid phosphatase 5b <t>(TRAP5b).</t> (B) Immunoblot analysis of unmodified Rap1a and GAPDH (loading control) using 30 μL of bone homogenate from long bones of mice; positive control = cell lysate from RAW264.7 osteoclasts (D5) treated with 400 nM RAM2061 for 48 h). (C) Quantification of RAM2061 accumulation in long bones and mandibles of CD-1 mice ( n = 5 per group, data are represented as mean ± SD). (D) MicroCT analysis portraying morphometric analysis of the proximal tibia specimens. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 5; zoledronic acid (ZA) n = 4; * denotes p <.05, ** denotes p <.01 per t-test). (E) The growth plate in the distal femur of tartrate resistant acid phosphatase (TRAP)-stained sections were identified and 1000 μm from the most distal area was used to encapsulate the area of interest. Quantification of osteoclast numbers in the defined area per femur was performed. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 4; ZA n = 4, * denotes p<.05, *** denotes p <.001 per t-test). (F) Representative images of TRAP-stained sections of distal femurs (scale bar = 50 μm). The arrows indicate TRAP+ osteoclasts (Tb = trabecular bone, BM = bone marrow).
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( A ) <t>TRAP</t> staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) <t>ELISA</t> analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .
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( A ) <t>TRAP</t> staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) <t>ELISA</t> analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .
Mouse Trap (Trap 5b) Elisa Kit, supplied by Immunodiagnostic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
mouse trap (trap 5b) elisa kit - by Bioz Stars, 2026-10
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( A ) <t>TRAP</t> staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) <t>ELISA</t> analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .
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( A ) <t>TRAP</t> staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) <t>ELISA</t> analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .
Mouse Trap Assay Kit Sb Tr103, supplied by Immunodiagnostic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Immunodiagnostic Systems mouse trap elisa kit
( A ) <t>TRAP</t> staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) <t>ELISA</t> analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .
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Fig. 1 Aged bone marrow-derived mesenchymal stem cells (BMMSCs) showed degenerative properties in osteogenesis and melatonin (MEL) promoted osteogenesis of aged BMMSCs. a) Haematoxylin and eosin (H&E) staining (40×) of proximal tibiae and b) micro-CT (μCT) imaging of distal femora of aged and young mice. c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). An independent-samples t-test was used to calculate the p-value in Figure 1c. d) Alkaline phosphatase (ALP) staining (20×) and e) Alizarin Red staining (20×) to analyze the osteogenesis of young and aged BMMSCs. f) Gene enrichment analysis of genes related to senile osteoporosis predicted using the Genecard database, DisGeNET database, and OMIM database, highlighting pathways where MEL metabolism and effects showed the highest enrichment in WikiPathways. Chi-squared test was used to calculate the p-value. g) MEL in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). h) ALP staining (5×), i) ALP level analysis, j) Alizarin Red staining (5×), and k) mineralization level analysis were performed. *p < 0.05, significant differences between each indicated group analyzed using one-way analysis of variance (ANOVA). CTR, control group; IL-18, interleukin-18; MAPK, mitogen-activated protein kinase; ns, not significant; PI3K-AKT, phosphoinositide 3-kinase-protein kinase B; VEGFA-VEGFR2, vascular endothelial growth factor-vascular endothelial growth factor receptor 2.

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.bjr-2024-0112.r2

Figure Lengend Snippet: Fig. 1 Aged bone marrow-derived mesenchymal stem cells (BMMSCs) showed degenerative properties in osteogenesis and melatonin (MEL) promoted osteogenesis of aged BMMSCs. a) Haematoxylin and eosin (H&E) staining (40×) of proximal tibiae and b) micro-CT (μCT) imaging of distal femora of aged and young mice. c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). An independent-samples t-test was used to calculate the p-value in Figure 1c. d) Alkaline phosphatase (ALP) staining (20×) and e) Alizarin Red staining (20×) to analyze the osteogenesis of young and aged BMMSCs. f) Gene enrichment analysis of genes related to senile osteoporosis predicted using the Genecard database, DisGeNET database, and OMIM database, highlighting pathways where MEL metabolism and effects showed the highest enrichment in WikiPathways. Chi-squared test was used to calculate the p-value. g) MEL in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). h) ALP staining (5×), i) ALP level analysis, j) Alizarin Red staining (5×), and k) mineralization level analysis were performed. *p < 0.05, significant differences between each indicated group analyzed using one-way analysis of variance (ANOVA). CTR, control group; IL-18, interleukin-18; MAPK, mitogen-activated protein kinase; ns, not significant; PI3K-AKT, phosphoinositide 3-kinase-protein kinase B; VEGFA-VEGFR2, vascular endothelial growth factor-vascular endothelial growth factor receptor 2.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Derivative Assay, Staining, Micro-CT, Imaging, Enzyme-linked Immunosorbent Assay, Control

Fig. 3 Inhibition of autophagy could counteract melatonin (MEL)-induced osteogenic promotion in aged mice. a) Micro-CT (μCT) imaging of distal femora and b) haematoxylin and eosin (H&E) staining (40×) of proximal tibiae of aged mice treated with MEL for six weeks (50 mg kg-1 body weight per day). c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp) were measured by μCT scanning. d) to f) Immunohistopathology (200×) showed the osteogenic-related protein presentations of d) matricellular molecular osteocalcin (OCN) and e) osteogenic marker Osterix in proximal tibia bone tissue, and f) integral optical density (IOD) was calculated. g) to i) Concentrations of g) bone resorption marker ACP5, h) osteogenic marker OCN, and i) MEL in serum were analyzed by enzyme-linked immunosorbent assay (ELISA). *p < 0.05, significant differences between each indicated group analyzed using Fisher’s exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; ns, not significant.

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.bjr-2024-0112.r2

Figure Lengend Snippet: Fig. 3 Inhibition of autophagy could counteract melatonin (MEL)-induced osteogenic promotion in aged mice. a) Micro-CT (μCT) imaging of distal femora and b) haematoxylin and eosin (H&E) staining (40×) of proximal tibiae of aged mice treated with MEL for six weeks (50 mg kg-1 body weight per day). c) Bone microstructure parameters such as bone mineral density (BMD), trabecular bone volume per total volume (BV/TV), trabecular bone thickness (Tb.Th), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp) were measured by μCT scanning. d) to f) Immunohistopathology (200×) showed the osteogenic-related protein presentations of d) matricellular molecular osteocalcin (OCN) and e) osteogenic marker Osterix in proximal tibia bone tissue, and f) integral optical density (IOD) was calculated. g) to i) Concentrations of g) bone resorption marker ACP5, h) osteogenic marker OCN, and i) MEL in serum were analyzed by enzyme-linked immunosorbent assay (ELISA). *p < 0.05, significant differences between each indicated group analyzed using Fisher’s exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; ns, not significant.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Inhibition, Micro-CT, Imaging, Staining, Marker, Enzyme-linked Immunosorbent Assay, Control

Fig. 5 Administration of melatonin (MEL) has therapeutic effects on bone fracture healing in aged mice. a) Representative radiograph and micro-CT (μCT) of femora in aged mice (n = 6 per group) four weeks after open femoral mid-shaft fracture. b) Nonunion frequency and c) μCT measurement of bone volume per total volume (BV/TV) and bone mineral density (BMD) in callus area of the fractured femora four weeks after open femoral mid-shaft fracture. d) Haematoxylin and eosin (H&E) staining (50×) of fractured femur. e) Ratio of newly formed bone, cartilage, and fibrous tissue area and quantification of the area of new bone formation. f) Immunohistopathology (200×) of fractured femur, showing the osteogenic-related protein matricellular protein osteocalcin (OCN) in the fracture callus. The integral optical density (IOD) of OCN was calculated. g) Osteoblast counts in the fracture callus were assessed by counting the number of osteoblasts on each H&E-stained section, with osteoblast number/bone perimeter (N.Ob/B.Pm) (/mm) determined using Image-Pro Plus software (Media Cybernetics, USA). Blue arrows indicate typical osteoblasts in the sections, highlighting their distribution in the fracture callus. h) Evaluation of osteoclasts in fracture callus. The numbers of osteoclasts were counted based on tartrate-resistant acid phosphatase (TRAP)-stained sections, and osteoblast number/total area (N.OC/T.Ar) (/mm2) was determined by Image-Pro Plus software. i) Protein presentations from bone marrow-derived mesenchymal stem cells (BMMSCs) extracted from tibiae after fracture healing of autophagy markers LC3BII, Beclin, and the osteogenesis marker OCN. *p < 0.05, significant differences between each indicated group analyzed using Fisher's exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; GelMA, methacrylated gelatin; ns, not significant.

Journal: Bone & Joint Research

Article Title: Melatonin alleviates senile osteoporosis by regulating autophagy and enhancing fracture healing in aged mice

doi: 10.1302/2046-3758.142.bjr-2024-0112.r2

Figure Lengend Snippet: Fig. 5 Administration of melatonin (MEL) has therapeutic effects on bone fracture healing in aged mice. a) Representative radiograph and micro-CT (μCT) of femora in aged mice (n = 6 per group) four weeks after open femoral mid-shaft fracture. b) Nonunion frequency and c) μCT measurement of bone volume per total volume (BV/TV) and bone mineral density (BMD) in callus area of the fractured femora four weeks after open femoral mid-shaft fracture. d) Haematoxylin and eosin (H&E) staining (50×) of fractured femur. e) Ratio of newly formed bone, cartilage, and fibrous tissue area and quantification of the area of new bone formation. f) Immunohistopathology (200×) of fractured femur, showing the osteogenic-related protein matricellular protein osteocalcin (OCN) in the fracture callus. The integral optical density (IOD) of OCN was calculated. g) Osteoblast counts in the fracture callus were assessed by counting the number of osteoblasts on each H&E-stained section, with osteoblast number/bone perimeter (N.Ob/B.Pm) (/mm) determined using Image-Pro Plus software (Media Cybernetics, USA). Blue arrows indicate typical osteoblasts in the sections, highlighting their distribution in the fracture callus. h) Evaluation of osteoclasts in fracture callus. The numbers of osteoclasts were counted based on tartrate-resistant acid phosphatase (TRAP)-stained sections, and osteoblast number/total area (N.OC/T.Ar) (/mm2) was determined by Image-Pro Plus software. i) Protein presentations from bone marrow-derived mesenchymal stem cells (BMMSCs) extracted from tibiae after fracture healing of autophagy markers LC3BII, Beclin, and the osteogenesis marker OCN. *p < 0.05, significant differences between each indicated group analyzed using Fisher's exact test, one-way analysis of variance (ANOVA), or Tukey’s post-hoc test. CTR, control group; GelMA, methacrylated gelatin; ns, not significant.

Article Snippet: The concentration of tartrate-resistant acid phosphatase 5 (ACP5), OCN, MEL, in mouse serum were measured with Mouse Tartrate Resistant ACP5 ELISA Kit, Mouse Osteocalcin ELISA Kit, and Mouse Melatonin ELISA Kit (Elabscience Biotechnology, China), respectively, according to the manufacturer’s instructions.

Techniques: Micro-CT, Staining, Software, Derivative Assay, Marker, Control

RAM2061 does not significantly impact normal bone remodeling in CD-1 mice. (A) Schematic representation of drug administration/dosing and downstream readouts conducted. Abbreviations: procollagen type 1 N-propeptide (P1NP); C-terminal telopeptide (CTX), tartrate resistant acid phosphatase 5b (TRAP5b). (B) Immunoblot analysis of unmodified Rap1a and GAPDH (loading control) using 30 μL of bone homogenate from long bones of mice; positive control = cell lysate from RAW264.7 osteoclasts (D5) treated with 400 nM RAM2061 for 48 h). (C) Quantification of RAM2061 accumulation in long bones and mandibles of CD-1 mice ( n = 5 per group, data are represented as mean ± SD). (D) MicroCT analysis portraying morphometric analysis of the proximal tibia specimens. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 5; zoledronic acid (ZA) n = 4; * denotes p <.05, ** denotes p <.01 per t-test). (E) The growth plate in the distal femur of tartrate resistant acid phosphatase (TRAP)-stained sections were identified and 1000 μm from the most distal area was used to encapsulate the area of interest. Quantification of osteoclast numbers in the defined area per femur was performed. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 4; ZA n = 4, * denotes p<.05, *** denotes p <.001 per t-test). (F) Representative images of TRAP-stained sections of distal femurs (scale bar = 50 μm). The arrows indicate TRAP+ osteoclasts (Tb = trabecular bone, BM = bone marrow).

Journal: JBMR Plus

Article Title: Geranylgeranyl diphosphate synthase inhibition impairs osteoclast differentiation, morphology, and resorptive activity

doi: 10.1093/jbmrpl/ziae133

Figure Lengend Snippet: RAM2061 does not significantly impact normal bone remodeling in CD-1 mice. (A) Schematic representation of drug administration/dosing and downstream readouts conducted. Abbreviations: procollagen type 1 N-propeptide (P1NP); C-terminal telopeptide (CTX), tartrate resistant acid phosphatase 5b (TRAP5b). (B) Immunoblot analysis of unmodified Rap1a and GAPDH (loading control) using 30 μL of bone homogenate from long bones of mice; positive control = cell lysate from RAW264.7 osteoclasts (D5) treated with 400 nM RAM2061 for 48 h). (C) Quantification of RAM2061 accumulation in long bones and mandibles of CD-1 mice ( n = 5 per group, data are represented as mean ± SD). (D) MicroCT analysis portraying morphometric analysis of the proximal tibia specimens. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 5; zoledronic acid (ZA) n = 4; * denotes p <.05, ** denotes p <.01 per t-test). (E) The growth plate in the distal femur of tartrate resistant acid phosphatase (TRAP)-stained sections were identified and 1000 μm from the most distal area was used to encapsulate the area of interest. Quantification of osteoclast numbers in the defined area per femur was performed. Individual data points are shown with mean ± SD (PBS n = 5; RAM2061 n = 4; ZA n = 4, * denotes p<.05, *** denotes p <.001 per t-test). (F) Representative images of TRAP-stained sections of distal femurs (scale bar = 50 μm). The arrows indicate TRAP+ osteoclasts (Tb = trabecular bone, BM = bone marrow).

Article Snippet: Commercial ELISA kits (Novus Biologics) were utilized for measuring plasma levels of TRAP5b (NBP2-76463), c-terminal telopeptide (CTX) (NBP2-82400), and procollagen type I N-propeptide (P1NP) (NBP2-76466).

Techniques: Western Blot, Control, Positive Control, Staining

( A ) TRAP staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) ELISA analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .

Journal: eLife

Article Title: Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and β-catenin-OPG/Jagged1 pathway

doi: 10.7554/eLife.98900

Figure Lengend Snippet: ( A ) TRAP staining (left) and histomorphometric analysis (right) of histological sections obtained from the metaphysis region of distal femurs from the 12-week-old male Malat1 f/f and Malat1 cKO Ocn littermate mice. n = 5–6/group. Oc.S/BS, osteoclast surface per bone surface; N.Oc/B.Pm, number of osteoclasts per bone perimeter. ( B ) Serum TRAP levels of 12-week-old male mice. ( C ) A schematic diagram (left) of the co-culture system with primary osteoblasts and bone marrow cells in trans-wells. TRAP staining (middle) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). (n =5 replicates from two experiments). ( D–E ) qPCR analysis of mRNA expression of Tnfrsf11b (encoding OPG) ( D ) and Tnfsf11 (encoding RANKL) ( E ) in calvarial osteoblasts (n =5/group). ( F ) The expression ratio of Rankl/Opg in calvarial osteoblasts. ( G ) ELISA analysis of OPG levels in the serum from the 12-week-old male WT and Malat1 -/- mice (n = 11–12/group). ( H ) Immunoblot analysis of OPG expression in the bone marrow supernatant from the 12-week-old male WT and Malat1 -/- mice. Bottom: Ponceau Staining of the gels showing an equivalent amount of total proteins loaded between samples. ( I ) Osteoclast differentiation of WT and Malat1 -/- bone marrows stimulated with RANKL (40 ng/ml) and M-CSF C.M. (1:20) with or without OPG (2.5 ng/ml) for five days. TRAP staining (left panel) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). TRAP-positive cells appear red in the photographs. n = 5 replicates. ( J ) Osteoclast differentiation of the cocultures of the indicated calvarial osteoblasts and WT bone marrow cells treated with 10 nM of VitD3 and 1 μM of prostaglandinE2 for 6 days in the presence or absence of OPG (1 ng/ml). TRAP staining (left) was performed and the number of TRAP-positive MNCs (≥3 nuclei/cell) per well was calculated (right panel). n = 3 replicates. Data are mean ± SD. A-G, *p < 0.05; **p < 0.01 by Student’s t test; I,J *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA with Bonferroni’s multiple comparisons test. ns, not statistically significant. Scale bars: A 200 µm; C,I,J 100 µm. Figure 5—source data 1. Malat1 promotes OPG expression in osteoblasts to suppress osteoclastogenesis. Figure 5—source data 2. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 3. Original files for western blot analysis displayed in .

Article Snippet: Commercial assay or kit , Mouse Tartrate Resistant Acid Phosphatase (TRAP) ELISA Kit , MyBioSource.com , MBS1601167 , .

Techniques: Staining, Co-Culture Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: eLife

Article Title: Long non-coding RNA Malat1 fine-tunes bone homeostasis and repair by orchestrating cellular crosstalk and β-catenin-OPG/Jagged1 pathway

doi: 10.7554/eLife.98900

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse Tartrate Resistant Acid Phosphatase (TRAP) ELISA Kit , MyBioSource.com , MBS1601167 , .

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reporter Assay, Software