After seeding for 24 h, cells were plated in triplicate and pre-treated for 30 min with four different conditions as mentioned earlier, followed by a 5-day incubation with 50 ng/mL soluble RANKL (462-TEC, R&D systems, Minneapolis, MN, USA) to induce osteoclastogenesis. Subsequently, cells were fixed in 4% paraformaldehyde for 10 min, and then stained for determination of TRAP activity. A commercial TRAP staining kit (Cosmo Bio, Tokyo, Japan) was used at 37 °C for 60 min to determine osteoclast formation. TRAP-positive multinucleated cells, which had more than 3 nuclei, were considered to denote achievement of osteoclast differentiation. The osteoclast number was finally determined using ImageJ software (NIH, MA, USA).
462 tec
Manufactured by R&D Systems
Sourced in United States
The 462-TEC is a laboratory instrument designed for temperature control. It features a compact and durable construction with a digital display that provides precise temperature readings. The device is intended for use in various scientific and research applications that require accurate temperature management.
Automatically generated - may contain errors
Lab products found in correlation
M csf, by R&D Systems (3 mentions)
Rlt buffer, by Qiagen (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Leukocyte acid phosphatase staining kit, by Merck Group (1 mentions)
C57bl 6j, by Jackson ImmunoResearch (1 mentions)
Suspension culture dish, by Corning (1 mentions)
Leukocyte acid phosphatase kit, by Merck Group (1 mentions)
L3012, by Merck Group (1 mentions)
Ct adus100, by InvivoGen (1 mentions)
Tlr nacga, by InvivoGen (1 mentions)
Tlrl patc, by Cayman Chemical (1 mentions)
Zoledronic acid, by Cayman Chemical (1 mentions)
Ctl 4112, by BioLegend (1 mentions)
Mem alpha medium, by Thermo Fisher Scientific (1 mentions)
Rtl buffer, by Qiagen (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
A8960, by Merck Group (1 mentions)
13 protocols using 462 tec
1
Osteoclast Differentiation Assay
2
Osteoclast Differentiation from Murine Bone Marrow
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Bone marrow cells from 8- to 12-wk-old Notumflox/flox and Runx2-creNotumflox/flox mice were cultured in suspension culture discs (Corning) in complete α-MEM with 30 ng/ml macrophage colony-stimulating factor (M-CSF) (416-ML-050; R&D Systems, Minneapolis, MN, USA) for 2 d, and the adherent bone marrow–derived macrophages (BMMs) were used as osteoclast progenitors (28 (link), 29 (link)). BMMs were detached and spot seeded in 24-well plates (40,000 cells/well) and cultured in complete α-MEM with 30 ng/ml M-CSF with or without the addition of 4 ng/ml receptor activator of nuclear factor κ-B ligand (RANKL) (462-TEC; R&D Systems) to induce osteoclast differentiation. The medium was changed after 3 d and cells harvested for RNA isolation by lysis in RLT buffer (Qiagen, Germantown, MD, USA) 4 d after seeding.
3
Osteoclast Differentiation Assay
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For the osteoclast differentiation assay, mouse bone marrow macrophages (BMMs) were harvested from bone marrow cells in the long bones of 8-week-old wild-type mice (C57BL/6J, The Jackson Laboratory, Sacramento, CA, USA). In brief, bone marrow cells were flushed out from the femurs and tibias, treated with red blood cell lysis buffer, and suspended in 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Corning, New York, NY, USA). Cells were cultured in the presence of M-CSF (10 ng/mL, R&D Systems, Minneapolis, MN, USA, 416-ML), and 1 day later, non-adherent cells were plated at a density of 0.5 × 106 cells/well in 24-well plates. BMMs were differentiated into osteoclasts by treating them with M-CSF (10 ng/mL) and RANKL (20 ng/mL, R&D Systems, 462-TEC), and two days later, liquid chlorobenzene was added daily at four concentrations (0 (control), 1, 10, and 100 µg/mL) for 14 days. To assess osteoclast differentiation, TRAP staining was performed using a leukocyte acid phosphatase staining kit (Sigma, 387A) according to the manufacturer’s protocol. The TRAP-stained osteoclasts were detected using an Evos microscope (Applied Biosystems, Waltham, MA, USA).
4
Osteoclast Differentiation in Bone Marrow Cells
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We cultured 1 x 105 bone marrow cells, isolated from 7–8-week-old WT, Dcstamp-/-, Tg(hTNF) and Dcstamp-/-;Tg(hTNF) mice, in a 96-well plates in alpha - DMEM media supplemented with 10 μg/mL of M-CSF (415-ML, R&D system, Minneapolis., MN) 2 mM L-glutamine, 100 μg/mL streptomycin, 1 mM of non-essential amino acids, 1 mM sodium pyruvate, and 0.5 μM of amphotericin B. On day three, fresh media containing 10 μg/mL of M-CSF and 10 μg/mL of RANKL was added (462- TEC, R&D system, Minneapolis., MN) and cell cultures were maintained for additional four days. Cells were then fixed with citrate/37% of formaldehyde solution. Osteoclast differentiation was evaluated with a TRAP activity kit according to the manufacturer’s instructions (387A, Sigma-Aldrich Co LLC. St Louis., MO).
5
Isolation and Culture of Murine Bone Marrow Macrophages
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Bone marrow cells were flushed from femurs and tibias from 8 to 12 week-old C57BL/6N mice, washed and cultured in complete α-MEM with 30 ng·mL−1 M-CSF (R&D, 416-ML-050) in a suspension culture dish (Corning Costar Ins., NY), to which stromal cells and lymphoid cells cannot adhere, at 37 °C for 2–3 days. Cells were washed vigorously with PBS to remove any non-adherent cells and then washed with cold (4 °C) 0.02% EDTA in PBS to release the attached bone marrow macrophages (BMM). BMM purity was demonstrated by expression of the monocyte marker CD11b/Mac-1 in all cells, whereas no cells expressed the T- and B-cells markers CD45R and CD3. Cells were spot seeded in 24-well plates (40 000 cells in 40 μL complete α-MEM). For osteoclast generation, cells were cultured in 30 ng·mL−1 M-CSF and 4 ng·mL−1 RANKL (R&D, 462-TEC) for 3–4 days.
6
Macrophage and Osteoclast Differentiation
7
Osteoclast Formation Assay with Exosomes
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For studies on osteoclast formation and differentiation, cells from the monocytic cell line RAW264.7 were seeded in 24 well plates at a density of 1,5x104 cells per well. After an initial attachment period of 24 hours, the cells were cultured in the absence (control) or presence of recombinant mouse RANKL (RANKL; 462-TEC, R&D Systems) at a concentration of 2 ng/mL, alone or in combination with exosomes for 96 h. Exosomes from either TRAMP-C1 tumor cells or MLg fibroblastic cells were added at two different concentrations, a higher concentration of 50 ng/103 seeded cells, or the lower concentration of 10 ng/103 seeded cells. After 96 h of culture, the cells were washed twice with PBS, fixed with ice-cold methanol for 10 minutes and stained for tartrate-resistant acid phosphatase (TRAP) using the Leukocyte Acid Phosphatase kit (Sigma-Aldrich) according to the manufacturer’s recommendations. Cells positive for TRAP with ≥ 3 nuclei were considered osteoclasts and counted.
8
Polarization of Bone Marrow-Derived Macrophages
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BMDMs were seeded in a six-well plate at an initial density of 5×105 cells per well and induced with 100 ng·mL−1 LPS (L3012, Sigma-Aldrich), 100 ng·mL−1 IFN-γ (285-IF, R&D System), LPS+IFN-γ, and RANKL (462-TEC, R&D Systems, Minneapolis, MN, USA) at different concentrations (1, 10, and 100 ng·mL−1) for 12 h. Polarized cells were harvested for mRNA analysis by reverse reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
9
Osteoclastogenesis Modulation by Curcuminoids
10
Macrophage and Osteoclast Differentiation
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Total bone marrow cells were harvested from femurs and tibias of 6-month-old Scarb1fl/fl and LysM-Cre;Scarb1fl/fl mice. After removing red blood cells with ACK buffer (0.1 mM EDTA, 10 mM KHCO3 and 150 mM NH4Cl, pH 7.2–7.3), bone marrow cells were plated in 10 cm tissue culture plates with 10 ml α-MEM complete media (10 % fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) supplemented with macrophage colony-stimulating factor (M-CSF; 30 ng/ml; cat. 216-MC, R&D Systems Minneapolis, MN). After 24 h of incubation, non-adherent cells were plated in Petri dishes (10 cm) and cultured with 10 ml α-MEM complete media supplemented with M-CSF (30 ng/ml) for 4 days. Those cells were used to culture macrophages and osteoclasts for analysis of mRNA and gDNA.
To obtain mRNA and genomic DNA, cells were then plated in 6-well plates at a density of 0.3 × 106 cells/well with 1.5 ml of α-MEM complete media supplemented with 30 ng/ml M-CSF for 3 days to obtain macrophages and with 30 ng/ml M-CSF and 30 ng/ml RANKL (RANKL cat. 462-TEC, R&D Systems, Minneapolis, MN, USA) for 3 days to obtain osteoclasts.
To obtain mRNA and genomic DNA, cells were then plated in 6-well plates at a density of 0.3 × 106 cells/well with 1.5 ml of α-MEM complete media supplemented with 30 ng/ml M-CSF for 3 days to obtain macrophages and with 30 ng/ml M-CSF and 30 ng/ml RANKL (RANKL cat. 462-TEC, R&D Systems, Minneapolis, MN, USA) for 3 days to obtain osteoclasts.
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