Synovial fibroblasts (passage 4) were seeded at 5 × 105 cells in 48-well plates and after 12 hours the cells were treated or not with PRL for 16 hours. Cyt were then added or not to the culture, and after 24 hours, bone marrow cells (2 × 106) containing osteoclast progenitors, harvested from the tibias and femurs of 8-week-old C57BL/6 mice, wild type (Prlr+/+) or null for the PRL receptor (Prlr-/-), were delivered in culture medium with 1, 25 dihydroxy-vitamin D3 (10-8 M, Sigma-Aldrich). Co-cultures were incubated for 10 days changing for new medium (containing 1, 25 dihydroxy-vitamin D3, +/- Cyt, +/- PRL) every 2 days. Co-cultures were fixed with 3.7% formaldehyde for 10 minutes at room temperature, washed twice with PBS, air-dried, and incubated for 20 minutes at 37 oC with TRAP activity staining mix. Osteoclastogenesis was assessed by counting the number of enlarged TRAP-positive (TRAP+) cells.
Dihydroxy vitamin d3
Manufactured by Merck Group
Sourced in United States, Germany
Dihydroxy-vitamin D3 is a chemical compound that is a metabolite of vitamin D3. It is a type of laboratory equipment used for research and analysis in various scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin mixture, by Merck Group (2 mentions)
Menadione, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
D2650, by Merck Group (1 mentions)
17β estradiol, by Cayman Chemical (1 mentions)
Hemin 51 280, by Merck Group (1 mentions)
A3059, by Merck Group (1 mentions)
Budesonide, by AstraZeneca (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
C11330500bt, by Thermo Fisher Scientific (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Pcs 500 012tm, by American Type Culture Collection (1 mentions)
Alizarin red solution, by Merck Group (1 mentions)
β glycerophosphate, by STEMCELL (1 mentions)
Ascorbat 2 phosphate, by Merck Group (1 mentions)
6 protocols using dihydroxy vitamin d3
1
Osteoclastogenesis Assay in Synovial Fibroblasts
2
Assay Reagent Preparation Protocol
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Budesonide (gift from AstraZeneca, Sweden), Org34517 (gift from Chiesi Farmaceutici, Parma, Italy), progesterone (57-83-0, Cayman Chemical), 17β-estradiol (50-28-2, Cayman Chemical), dexamethasone (D8893), triiodo-l-thyronine (T2877), retinoic acid (R2625), 9-cis-retinoic acid (R4643), 7β-hydroxycholesterol (H6891), and hemin (51280) (all from Sigma-Aldrich) were dissolved in dimethyl sulphoxide (DMSO) (D2650, Sigma-Aldrich) as stocks of 1 to 10 mM. Final DMSO concentrations on cells were ≤0.1%. Dihydroxy vitamin D3 (D1530, Sigma-Aldrich) was dissolved in absolute ethanol as a stock of 10 mM. Recombinant human IL1B (201-LB, R&D Systems) was dissolved in phosphate-buffered saline (PBS) (14190144, Thermo) containing 0.1% bovine serum albumin (BSA) (A3059, Sigma-Aldrich). G418 (A1720, Sigma-Aldrich) was dissolved in sterile water as stocks of 100 mg/ml.
3
Osteogenesis Evaluation of Fetal Osteoblasts
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To assess osteogenesis, the differentiation capacity of human fetal osteoblasts (hFOB 1.19 cells), a preosteoblastic cell line, was assessed as previously described. Briefly, for proliferation of hFOB 1.19 cells, cells were cultured at 33.5°C with 5% CO2 to 80% confluence in standard DMEM:F12 media supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD) and 1% Penicillin/Streptomycin mixture (Sigma, St. Louis, MO). To induce differentiation of the hFOB 1.19 cells, the standard media was supplemented with 100 μg/ml ascorbic acid, 10−8 M menadione, and 10−8 M dihydroxy‐vitamin D3 (Sigma, St. Louis, MO), and the cells were cultured at 39.5°C with 5% CO2 until the desired timepoint. In some experiments the FAK inhibitor, PF‐573228, which is known to inhibit focal adhesion turnover in vitro,16 was suspended in dimethyl sulfoxide (DMSO), and added to differentiation media at a molar concentration of 200 nM. This concentration was determined based on levels of FAK phosphorylation activity at varying FAK inhibitor concentrations (Figure S1 ). In addition, an identical amount of DMSO without FAK inhibitor was added to control media. Media was changed every 3 days unless otherwise stated for all experimental methods.
4
Osteogenic Differentiation of Human Fetal Osteoblasts
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To assess osteogenesis, the differentiation capacity of human fetal osteoblasts (hFOB 1.19 cells), a preosteoblastic cell line, was assessed as previously described (Lim and Donahue, 2007 (link)). Briefly, for proliferation of hFOB 1.19 cells, cells were cultured at 33.5°C with 5% CO2 to 80% confluence in standard DMEM:F12 media supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD) and 1% Penicillin/Streptomycin mixture (Sigma, St. Louis, MO). To induce differentiation of the hFOB 1.19 cells, the standard media was supplemented with 100 μg/mL ascorbic acid, 10−8 M menadione, and 10−8 M dihydroxy-vitamin D3 (Sigma, St. Louis, MO), and the cells were cultured at 39.5°C with 5% CO2 until the desired timepoint. Media was changed every 3 days unless otherwise stated for all experimental methods. All substrates used were sterilized by completely submerging the substrates in 70% ethanol for 5 min before the ethanol was aspirated and remaining ethanol allowed to evaporate for 1 h under UV light.
5
Osteogenic Differentiation of BMSCs
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Human bone marrow-derived mesenchymal stem cells (BMSCs) were purchased from ATCC (ATCCⓇ PCS-500-012TM, Manasas, VA, USA) and maintained in DMEM/F12 medium (C11330500BT, Gibco，Grand Island, NY, USA ) containing 10% FBS (10099-141，Gibco) in an incubator (37℃, 5% CO2).
For osteogenic differentiation of BMSCs, briefly, the cells were maintained in a specific medium for 14 days (d), and the medium was changed every 3 d. Specific culture medium was prepared (6 (link)) by supplementing DMEM/F12 with 1% HyClone serum (16050-122, Gibco), 200 μM L-glutamine (25030-081, Gibco), 100 nM dexamethasone (D4902, purity≥97%, Sigma aldrich, St. Louis, MO, USA), 10 mM β-glycerolphosphate (50020, purity≥98%, Sigma aldrich), 10 nM dihydroxyvitamin D3 (50020, purity≥99%, Sigma aldrich) and 80 μg/ml L-Ascorbic acid (A7506, Sigma aldrich).
For osteogenic differentiation of BMSCs, briefly, the cells were maintained in a specific medium for 14 days (d), and the medium was changed every 3 d. Specific culture medium was prepared (6 (link)) by supplementing DMEM/F12 with 1% HyClone serum (16050-122, Gibco), 200 μM L-glutamine (25030-081, Gibco), 100 nM dexamethasone (D4902, purity≥97%, Sigma aldrich, St. Louis, MO, USA), 10 mM β-glycerolphosphate (50020, purity≥98%, Sigma aldrich), 10 nM dihydroxyvitamin D3 (50020, purity≥99%, Sigma aldrich) and 80 μg/ml L-Ascorbic acid (A7506, Sigma aldrich).
6
Osteogenic Differentiation of Microtissue-Derived Cells
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For osteogenic differentiation, cells derived from microtissue-SVF after 1 week in culture were seeded at a density of 1.1 × 10 3 /cm 2 in EGM-2 and incubated overnight. On the next day, medium was changed to osteogenic differentiation medium DMEM-low glucose containing 10 % FCS, 2 mM L-glutamine, 100 U/mL P/S, 10 nM dexamethasone, 150 µM ascorbat-2-phosphate (Sigma-Aldrich), 10 mM β-glycerophosphate (StemCell Technologies, Cologne, Germany) and 10 nM dihydroxy-vitamin D3 (Sigma-Aldrich) or control medium consisting of DMEM : F12/L-glutamine with 10 % FCS and 100 U/ mL P/S. Medium was changed every 3-4 d. After 21 d, osteogenic differentiation was analysed with alizarin red staining and quantification through RT-PCR of the specific markers alkaline phosphatase (ALPL), osteocalcin (BGLAP) and osteopontin (SPP1). For alizarin red staining of calcified structures, cells were fixed for 1 h with 70 % ethanol at -20 °C and stained with alizarin red solution (Merck) for 15 min. Images were acquired using a light microscope (Axiovert 200). www.ecmjournal.org
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