Ihh promoter sequences were analyzed for putative Runx binding sites with PROMO3.0 (http://alggen.lsi.upc.es/ ) using version 8.3 of the TRANSFAC database. The promoter region (−) and (+) of the mouse Ihh gene was amplified by PCR from a murine Ihh BAC clone (CH29–567C21; CHORI). Primer sequences are available in Table S3 . Then the promoter regions were inserted into the pGL3-basic vector to construct the pGL3-Ihh promoter fragments. ATDC5 cells (Sigma) were cultured in 24-well plates, were transfected with the DNA mixture containing different pGL3-Ihh construct (0.3μg) and β-GAL-expressing plasmids (0.06 μg), with or without Runx1 expressing vector (psport6-CMV-Runx1, 0.3μg) using a calcium phosphate co-precipitation method. Luciferase was detected using Glo Luciferase Assay System (Promega) as described [20 (link), 22 (link)]. The β-GAL activity of the cell lysates was analyzed using β-Galactosidase Enzyme Assay System (E2000; Promega). The level of luciferase activity was normalized to the level of β-GAL activity.
Atdc5 cells
Manufactured by Merck Group
Sourced in United States
ATDC5 cells are a type of pre-adipocyte cell line derived from mouse embryonic carcinoma cells. They are commonly used in research as an in vitro model for the study of adipocyte differentiation and function.
Automatically generated - may contain errors
Lab products found in correlation
Ascorbic acid, by Merck Group (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Its premix, by Corning (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glo luciferase assay system, by Promega (1 mentions)
β galactosidase enzyme assay system, by Promega (1 mentions)
Collagenase, by Merck Group (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Dmem f12, by Merck Group (1 mentions)
A8960, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by R&D Systems (1 mentions)
Ls004176, by Roche (1 mentions)
α mem medium, by R&D Systems (1 mentions)
α mem medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
6 protocols using atdc5 cells
1
Runx1 Regulation of Indian Hedgehog Promoter
2
Culturing Murine Chondrogenic Cells
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Murine chondrogenic ATDC5 cells (Sigma-Aldrich; 99072806) were cultured in DMEM:F12 (Gibco, Billings, MT, USA; 11320-033) with 5% FCS (Gibco; 10437-028) and 100 U/mL penicillin and streptomycin (Gibco; 15140122) at 37 °C, 5% CO2 and 95% relative humidity.
Primary murine chondrocytes were isolated from rib cage cartilage of 10-day-old mice by collagenase (Sigma-Aldrich; C9891) digestion in DMEM:F12 and cultured in DMEM:F12 with 10% FCS and 50 μg/mL ascorbic acid at 37 °C, 5% CO2 5% O2 and 95% relative humidity.
Primary murine chondrocytes were isolated from rib cage cartilage of 10-day-old mice by collagenase (Sigma-Aldrich; C9891) digestion in DMEM:F12 and cultured in DMEM:F12 with 10% FCS and 50 μg/mL ascorbic acid at 37 °C, 5% CO2 5% O2 and 95% relative humidity.
3
Isolation and Culture of Osteoblasts, Osteoclasts, and Chondrogenic Cells
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The chondrogenic ATDC5 cells were purchased from Sigma and cultured in DMEM/Ham’s F12 medium supplemented with 2% FBS, 2 mM l -glutamine and 1% penicillin/streptomycin. In addition, primary osteoprogenitors (COB) were isolated from calvaria of 5-day-old wild-type neonates (C57BL/6 J) using Collagenase type II (50 mg/ml, Worthington, LS004176) and Dispase II (100 mg/ml, Roche, 10165859001) and were maintained in α-MEM medium (Gibco) containing 10% FBS (Gibco), 2 mM l -glutamine (Corning), 1% penicillin/streptomycin (Corning), and 1% nonessential amino acids (Corning). COBs were differentiated with ascorbic acid (200 uM, Sigma, A8960) and β-glycerophosphate (10 mM, Sigma, G9422). Finally, bone marrow cells were flushed from the femurs and tibias of 2-month-old mice (C57BL/6 J), and cultured in petri dishes in α-MEM medium with 10% FBS and 20 ng/ml of M-CSF (R&D systems) to obtain bone BM-OCP. After 12 h, nonadherent cells were replated into tissue culture dishes and cultured in the same medium for 3 days. BM-OCPs then differentiated into osteoclasts in the presence of RAMKL (20 ng/ml; R&D systems) and M-CSF (20 ng/ml; R&D systems) for 6 days.
4
ATDC5 Chondrocyte Maturation and IL-6 Response
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ATDC5 cells (Sigma, 99072806) were maintained in DMEM/F-12 (1:1) medium (Gibco, 11330032) supplemented with 5% FBS and 1% penicillin/streptomycin. ATDC5 cells were maturated in DMEM/F-12 (1:1) medium supplemented with 5% FBS, 1% penicillin/streptomycin, 1% ITS premix (Corning, 354352), 50μg/ml ascorbic acid, 10nM dexamethasone, and 10ng/ml TGF-β3 (Sigma, SRP6552) for 5, 10, and 15 days.
Both wild type and Adgrg6 KO ATDC5 cells were treated with 100ng/ml recombinant human IL-6 protein (rIL-6) (R&D System, 206-IL) for 2 hours before protein extraction. Both wild type and Adgrg6 KO ATDC5 cells were treated with 10nM Stattic in maturation medium for 10 days before RNA extraction.
Both wild type and Adgrg6 KO ATDC5 cells were treated with 100ng/ml recombinant human IL-6 protein (rIL-6) (R&D System, 206-IL) for 2 hours before protein extraction. Both wild type and Adgrg6 KO ATDC5 cells were treated with 10nM Stattic in maturation medium for 10 days before RNA extraction.
5
ATDC5 Chondrogenic Differentiation Protocol
6
ATDC5 Chondrogenic Differentiation Protocol
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Mouse chondrogenic ATDC5 cells (Sigma Aldrich) were grown in a proliferation medium (DMEM/F-12, GlutaMAX supplement (Thermo Fisher Scientific), 5% fetal bovine serum (Biowest), and 0.5 % Pen-Strep (Invitrogen)), under a humidified atmosphere at 37 °C, 5% CO2. Upon reaching confluency, cells were harvested and plated at 6400 cells·cm-2. Chondrogenic differentiation was induced by replacing the proliferation medium with a differentiation medium (proliferation medium supplemented with 10 μg mL−1 insulin (Sigma-Aldrich), 10 μg·mL−1 transferrin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 50 μg mL−1 ascorbic acid (Sigma-Aldrich)) and by adding BMP-2 and Noggin at various concentrations. In these experiments, the differentiation medium was refreshed every 3 days.
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