Bone marrow monocytes (BMMs) isolated from rat femurs were induced to differentiate towards osteoclasts by incubation in α‐MEM media supplemented with 50 ng/mL receptor activator of nuclear factor‐kappa B ligand (RANKL) (PeproTech, London, UK) and 50 ng/mL macrophage colony‐stimulating factor（M‐CSF）in the presence or absence of isoproterenol (1 μM). Osteoclast differentiation was confirmed using the Tartrate‐Resistant Acid Phosphatase Kit (Sigma‐Aldrich) as described in section 2.9. Formation of F‐actin rings, a marker of osteoclasts, was detected using rhodamine‐phalloidin fluorescent staining (Cytoskeleton, Denver, CO, USA); nuclei were counterstained with DAPI（Sigma‐Aldrich Co., Taufkirchen, Germany. To detect intracellular reactive oxygen species (ROS), BMMs cultured in conditioned or control media with isoprenaline were measured with the fluorescent oxidation‐sensitive probe 2’,7’‐dichlorodihydrofluorescein diacetate (DCFH‐DA; Genmed, Shanghai, China). Expressions of osteoclast‐related factors tartrate‐resistant acid phosphatase (Trap) and cathepsin k (Ctsk) were also determined by Western blotting using standard techniques and incubated anti‐Trap (ab96372, Abcam), anti‐Ctsk (ab19027, Abcam) and anti‐β‐tublin (ab6046, Abcam).
Tartrate resistant acid phosphatase kit
Manufactured by Merck Group
Sourced in United States
The Tartrate Resistant Acid Phosphatase (TRAP) Kit is a laboratory tool designed to detect and quantify TRAP activity. TRAP is an enzyme that serves as a biomarker for certain cellular processes. The kit provides the necessary reagents and protocols to measure TRAP levels in various sample types.
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Lab products found in correlation
Ab6046, by Abcam (1 mentions)
Ab19027, by Abcam (1 mentions)
Dcfh da, by Genmed Scientifics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Ab96372, by Abcam (1 mentions)
Sc52950, by Santa Cruz Biotechnology (1 mentions)
Murine macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Dentin discs, by Immunodiagnostic Systems (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
α mem medium, by Merck Group (1 mentions)
Evos xl inverted microscope, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Sangon (1 mentions)
Evos fl auto cell imaging system, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Positive selection, by Miltenyi Biotec (1 mentions)
Crosslaps for culture ctx 1 elisa, by Immunodiagnostic Systems (1 mentions)
Tritc conjugated phalloidin, by Merck Group (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
9 protocols using tartrate resistant acid phosphatase kit
1
Modulation of Osteoclast Differentiation by Isoproterenol
2
Quantification of Osteoclast Formation
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TRAP staining was performed with the tartrate-resistant acid phosphatase kit (Sigma 387A). Osteoclast numbers were assessed as multinucleated TRAP+ cells in each field of view.
3
Maxillary Molar Bone Remodeling
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Following a predetermined schedule, the maxillary bone containing the maxillary first molar was dissected on days 1, 3, and 7. The specimens were immediately immersed in 4% paraformaldehyde for 48 h, then demineralized in 10% EDTA (pH = 7.4) for 6 weeks. After paraffin embedding, the specimens were sectioned (4 μm) in the mesial-distal direction of the first molar and prepared for immunohistochemical staining and TRAP staining.
The slides were deparaffinized, treated with 0.125% trypsin and 5 µg/ml proteinase K solution for 20 min at 37 °C for antigen retrieval, and treated with 3% hydrogen peroxide for 20 min at room temperature. The sections were then blocked with goat serum for 1 h at room temperature, and incubated with antibodies to RANKL (1:200; sc-52,950, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. They were then incubated with secondary antibody for 20 min. After washing with phosphate-buffered saline, the sections were developed with diaminobenzidine. Ultimately, hematoxylin was used for counterstaining.
TRAP staining was performed with a tartrate-resistant acid phosphatase kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin. Cells containing more than 3 nuclei were considered osteoclasts.
The slides were deparaffinized, treated with 0.125% trypsin and 5 µg/ml proteinase K solution for 20 min at 37 °C for antigen retrieval, and treated with 3% hydrogen peroxide for 20 min at room temperature. The sections were then blocked with goat serum for 1 h at room temperature, and incubated with antibodies to RANKL (1:200; sc-52,950, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C. They were then incubated with secondary antibody for 20 min. After washing with phosphate-buffered saline, the sections were developed with diaminobenzidine. Ultimately, hematoxylin was used for counterstaining.
TRAP staining was performed with a tartrate-resistant acid phosphatase kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin. Cells containing more than 3 nuclei were considered osteoclasts.
4
Murine Osteoclast Precursor Differentiation
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Murine osteoclast precursors from 8 week-old C57BL/6 male mice were cultured in 96 well plates (1 × 105 mL−1, 200 μL per well) for 3 days co-cultured with 20 ng mL−1 murine CSF-1 and 300 ng mL−1 sRANKL with or without treatment of designed PDCs and M19 at the indicated concentration (0, 1.25, 2.5, 5 and 10 μM). Then, the fixed cells were stained using tartrate-resistant acid phosphatase kit (Sigma). TRAP-positive multinucleated cells were observed by a microscope (OLYMPUS-BX53).
5
In vitro Osteoclastogenesis Assay
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In vitro osteoclastogenesis was induced following standard protocols, as previously reported (Menale et al., 2019 (link)). Briefly, 4 × 105 splenocytes/well from NSG and NSG oc/oc littermates were cultured in 96-well plate in αMEM medium (Sigma-Aldrich) supplemented with 10% FBS, 1% penicillin/streptomycin (P/S ) and 1% glutamine in the presence of 25 ng/ml murine Macrophage-Colony Stimulating Factor (M-CSF ), 5 ng/ml human Transforming Growth Factor β1 (TGFβ1; both reagents from Peprotech, London, UK), 1 μM Dexamethasone (Sigma-Aldrich) and with or without 100 ng/ml murine Receptor Activator of Nf-kB Ligand (RANKL ) for 6 days. Mature osteoclasts were stained using the Tartrate Resistant Acid Phosphatase (TRAP) Kit (Sigma-Aldrich) following the manufacturer's instruction.
The same culture conditions were applied to achieve osteoclast formation on dentin discs (Immunodiagnostic Systems, Ltd., Scottsdale, AZ) and evaluate their resorptive capacity. After 3 weeks, dentin discs were rinsed with water, scraped to remove attached cells, stained with 1% Toluidine blue solution for 3 min and then washed with water to visualize resorption pits. Images were acquired on an EVOS XL Inverted Microscope (Thermo Fisher Scientific) for both TRAP+ mature osteoclasts and Toluidine blue-stained dentin discs.
The same culture conditions were applied to achieve osteoclast formation on dentin discs (Immunodiagnostic Systems, Ltd., Scottsdale, AZ) and evaluate their resorptive capacity. After 3 weeks, dentin discs were rinsed with water, scraped to remove attached cells, stained with 1% Toluidine blue solution for 3 min and then washed with water to visualize resorption pits. Images were acquired on an EVOS XL Inverted Microscope (Thermo Fisher Scientific) for both TRAP+ mature osteoclasts and Toluidine blue-stained dentin discs.
6
Synthesis and Characterization of M54 Compound
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Reagent M54 (M54, Figure 1A ) was supplied by Prof. Hu from the School of Pharmacy, Second Military Medical University. Briefly, sophocarpine was transformed into thiosophocarpine in high yield by treatment with Lawesson’s reagent. Then, M19 was synthesized by reaction of thiosophocarpine with NH2CH3. M54 was synthesized by reaction of M19 with CO2 and benzyl bromide. M54 was dissolved in ddH2O for use in vitro and dissolved in normal saline as vehicle for use in vivo. DMEM and fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, United States). Penicillin–streptomycin was obtained from Sangon Biotech (Shanghai City, IL, United States). Soluble recombinant mouse RANKL and M-CSF were purchased from R&D (R&D Systems, Minneapolis, MN, United States). Tartrate-resistant acid phosphatase (TRAP) Kit was obtained from Sigma-Aldrich (St. Louis, MO, United States). RAW264.7 cells were purchased from Central Plains Cell Institute (Shanghai, China).
7
Osteoclast Generation and Activity Assay
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Peripheral blood mononuclear cells (PBMCs) from healthy donors, obtained from the Scottish National Blood Transfusion Service (approved by Glasgow NHS Trust-East Ethics Committee), were isolated via Ficoll-paque PLUS (GE Healthcare) density gradient centrifugation and CD14+ monocytes isolated using positive selection (Miltenyi). Monocytes were cultured in selective survival media (Additional file 1 : Table S1). Osteoclasts were generated using supplementation with1ng/ml RANKL over 72 h before stimulation with vehicle, 10 ng/ml TNFα or 1000 ng/ml corticosterone (or DMSO vehicle) as appropriate. Osteoclast numbers were assessed by staining with tartrate-resistant acid phosphatase (TRAP) kit (Sigma-Aldrich). Osteoclast activity was assessed on mineral-coated plates (Corning) at day 14. Images were acquired using EVOS FL Auto Cell Imaging System (Life Technologies). Osteoclasts were identified as TRAP +ve multinucleated cells (nuclei ≥ 3). Resorption area was calculated using Fiji software (ImageJ) and defined as % resorbed area of entire well.
8
Osteoclast Characterization and Bone Resorption Assay
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Osteoclasts cultured on plastic were stained using the Tartrate Resistant Acid Phosphatase (TRAP) Kit (Sigma-Aldrich), following the manufacturer's instructions. Osteoclasts differentiated on bone slices were stained using alendronate conjugated to Alexa Fluor 488,24 (link) TRITC-conjugated phalloidin (Sigma-Aldrich) and TO-PRO-3 (Thermo Fisher Scientific). To evaluate bone resorption, the same bone slices were used for toluidine blue staining as previously described.25 (link) TRAP and toluidine blue images were acquired on a Zeiss AxioImager M2m microscope, while immunofluorescence staining images were acquired on a Leica TCS SP5 Laser Scanning Confocal microscope.
Quantification of CTX-I was performed on culture supernatants using CrossLaps for Culture (CTX-I) ELISA (Immunodiagnostic Systems), according to the manufacturer’s instructions.
Quantification of CTX-I was performed on culture supernatants using CrossLaps for Culture (CTX-I) ELISA (Immunodiagnostic Systems), according to the manufacturer’s instructions.
9
Quantitative Histological Analysis of Mouse Joint
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Formalin-fixed, EDTA-decalcified, paraffin-embedded mouse joint tissue specimens were sectioned and stained with H&E, toluidine blue (TB), or Tartrate-Resistant Acid Phosphatase (TRAP) Kit (Sigma-Aldrich). H&E and TB were semiquantitatively blindly evaluated for synovial inflammation/hyperplasia (scale of 0–5) and cartilage erosion (scale of 0–5) based on an adjusted, previously described method (83 (link)). TRAP staining was performed to measure number of osteoclasts using ImageJ software (NIH) on images acquired with a Nikon microscope, equipped with a QImaging digital camera.
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