Mineral resorption pit assay was used to evaluate the osteoclast activity as previously described.28 (link) Briefly, BMMs were initially seeded on 96-well Corning Osteo Assay Surface Plates (Corning, Tewksbury, MA, USA) at a seeding density of 2 × 104 per well. Subsequently, osteoclast differentiation was driven by CMG14–12 supernatant and 40 ng·mL−1 RANKL. Once we observed mature osteoclasts, the cells on the plate were removed by adding 10% sodium hypochlorite solution. After twice PBS washing, von Kossa staining was performed to visualize resorptive pits. Light microscopic images were captured for each well and pit areas were quantified by Image J (National Institutes of Health, Bethesda, MD, USA).
Osteo assay surface plate
Manufactured by Corning
Sourced in United States
Osteo Assay Surface plates are a type of cell culture labware designed for the study of bone-forming cells. They provide a specialized surface that supports the growth and differentiation of osteoblasts, the cells responsible for bone formation. These plates are a tool used in various research applications related to bone biology and development.
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Lab products found in correlation
Collagenase, by Merck Group (5 mentions)
α mem, by Thermo Fisher Scientific (4 mentions)
Cytation 3 cell imaging reader, by Agilent Technologies (3 mentions)
Image pro plus, by Media Cybernetics (3 mentions)
β glycerophosphate, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Axio observer a1, by Zeiss (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Merck Group (2 mentions)
Aqua bidest, by Merck Group (2 mentions)
L ascorbate, by Merck Group (2 mentions)
Alizarin red, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cellmatrix type 1 a, by Nitta Gelatin (2 mentions)
Leukocyte acid phosphatase kit, by Merck Group (2 mentions)
Dmi4000, by Leica camera (1 mentions)
Evos xl core microscope, by Thermo Fisher Scientific (1 mentions)
Ab92964, by Abcam (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Axio observer 5, by Zeiss (1 mentions)
86 protocols using osteo assay surface plate
1
Quantifying Osteoclast Resorptive Activity
2
Osteoclast Differentiation and Resorption Assay
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M-CSF-dependent BMMs were incubated in six-well plates (20 × 10 5 cells/well). After 24 h, the cells were supplied with stimulated by RANKL (100 ng/mL), M-CSF (33.3 ng/mL) until OC formation was observed.
The OCs (1 × 10 4 cells/well ) were then incubated on Corning Osteo Assay Surface plates (Corning, Inc.) and cultured with M-CSF (33.3 ng/mL), RANKL (100 ng/mL)and LY900009 (0, 100, 200, and 400 nM) for four days. A BioTek Cytation 3 Cell Imaging Reader (BioTek, Winooski, VT) and Image J software were used to photograph and analyze the total resorption pits.
The OCs (1 × 10 4 cells/well ) were then incubated on Corning Osteo Assay Surface plates (Corning, Inc.) and cultured with M-CSF (33.3 ng/mL), RANKL (100 ng/mL)and LY900009 (0, 100, 200, and 400 nM) for four days. A BioTek Cytation 3 Cell Imaging Reader (BioTek, Winooski, VT) and Image J software were used to photograph and analyze the total resorption pits.
3
Osteoclast Formation and Bone Resorption
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The BMMs (2 × 104) were seeded onto Corning Osteo Assay Surface plates (Corning Incorporated Life Science, NY, United States). The BMMs were stimulated with RANKL for 3 days. Then, different concentrations of BS (0.01, 0.1, and 1 μM) were supplemented until the osteoclasts were formed. All discs were washed with 5% sodium hypochlorite for 5 min followed by PBS for 10 min. Images were taken by light microscopy, and then the resorption pits were quantified by ImageJ.
4
Inhibition of Osteoclast Resorption
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M-CSF-dependent BMMs were incubated in six-well plates (20 × 105 cells/well). After 24 h, the cells were stimulated with RANKL (100 ng/mL), M-CSF (33.3 ng/mL) until OC formation was observed. The OCs (1 × 104 cells/well ) were then incubated on Corning Osteo Assay Surface plates (Corning, Inc.) and cultured with M-CSF (33.3 ng/mL), RANKL (100 ng/mL), and RO4929097 (0, 100, 200, and 400 nM) for four days. A BioTek Cytation 3 Cell Imaging Reader (BioTek, Winooski, VT) and Image J software were used to photograph and analyze the total resorption pits.
5
Osteoclastogenesis Assay with Activin-A
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CD14+ cells were isolated from blood from 4 healthy donors as described in the CD14+ cell isolation section. Osteoclastogenesis was performed on 96 well-Osteo Assay surface plates (Corning Costar, Lowell, MA, USA) as described in the osteoclastogenesis section either without or with Actvin-A and with both Activin-A and Follistatin. Each experimental condition was plated in quadruplicate. Cells were cultured for 14 and 21 days. To visualize the lysis of the calcium phosphate coating, wells were incubated for 5 min in 10% bleach, washed with H2O and air dried. For the quantification of the total resorbed area, 4 pictures were taken per well at 10 x magnification. Resorbed area was measured using Image-Pro Plus (MediaCybernetics, Rockville, USA) and depicted as percentage of resorption per well.
6
Quantifying Monocyte-Mediated Bone Resorption
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CD14+ monocytes from the two healthy volunteers were seeded (6.4 or 10 × 104 cells/well) in 96 well Osteo Assay surface plates (Corning costar, Lowell, MA, USA) and cultured for 2 weeks in the media described in Table 2 . Subsequently, cells were incubated with 10% bleach for 5 min, washed with H2O, and then air dried. For each well, 4 microscopic images (4× magnification) were taken with a Leica Ti Eclipse (Leica) to image the total well. Resorption was quantified as white area (resorbed Calcium-phosphate)/brown area (remaining Calcium-phosphate) × 100% and normalized to resorption of calcium phosphate in the control medium using ImageJ software (ImageJ, Bethesda, MD, USA).
7
Evaluating DZNep's Impact on Osteoclast Function
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To explore the effect of DZNep on the function of osteoclast, BMMs on complete α-MEM were seeded at the density of 1 × 104 cells/well, in triplicate, on the Osteo Assay Surface plates (Corning, NY, USA). After thirty-six hours, the culture medium were replaced by complete α-MEM, with RANKL (50 ng/mL), and various doses of DZNep (control, 6.25, 12.5, and 25 nM). For EZH2 gene knockdown studies, the cells were transfected with virus constructed of different EZH2 short hairpin RNA (shRNA) and negative control shRNA by mixed with polybrene ( nal concentration of 10ug/ml) for 48 hours. The transfection mixture was then replaced by complete α-MEM added with puromycin for 24 hours and the survived BMMs were seeded into a 96-well plate, in triplicate, at a density of 1 × 104 cells/well. The medium need be constantly replaced until the osteoclast had matured for 1 day. The 5% sodium hypochlorite were used to wash the wells for 3 min to remove the cells. The total area of resorption was photographed, and then calculated by Image J software (NIH, Bethesda, MD, USA).
8
Osteoclast Pit Formation Assay
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Osteo Assay Surface Plates (Corning, MA) were used for the pit formation assay as described previously50 (link). Briefly, BMMs (2 × 104 cells) were seeded onto the plate and cultured in α-MEM containing 10% FBS, M-CSF (30 ng/mL), and RANKL (100 ng/mL) for 5 days. BMMs were then treated with either AaE, artemisinin, artemisinic acid, or arteannuin B at the indicated concentrations for an additional 2 days. The cells were lysed with 5% sodium hypochlorite solution. The images of the resorption pits were obtained using light microscopy. The resorption pit areas were measured using ImageJ software (Ver. 1.6, NIH, Bethesda, MD).
9
Osteoclast Resorption Assay
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Mononuclear BMMs were plated on 24-well Corning Osteoassay surface plates at a density of 100,000 cells per well and fed as described above. At day 4–6 cells were washed away with 10% bleach as per manufacturer’s recommendations. Wells were imaged by light microscopy, and demineralization measurements were determined using NIH ImageJ.
10
Quantifying Osteoclast Resorption Activity
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Primary bone marrow macrophages were plated on Osteo Assay Surface plates (Corning) at a density of 1 × 105 cells per well. Cells were allowed to fully differentiate in the presence of 1.0% CMG 14–12 conditioned media and RANKL. The medium was completely removed on day 5 and 100 μL/well of 10% bleach or TRAP stain was added and allowed to incubate at room temperature for 5 minutes. The bleach solution or TRAP solution was then aspirated and the wells were washed twice with 150 μL of dH2O. The plate was then allowed to air dry completely at room temperature for 3–5 hours. The wells were observed under 4x objective for the formation of resorption pits and images were captured with light microscopy. Images were measured and analyzed using NIH Image J.
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