Non-confluent culture of RAW 264.7 osteoclast precursor (RAW) cell line (from ATCC) was harvested and cultured with minimum essential medium eagle alpha modification (alpha-MEM) (Sigma, St. Louis, MO, USA) supplemented with 2.2 G.L−1 NaHCO3 (Sigma), 15% fetal calf serum (Altana, Lawrenceville, GA, USA), 1% penicillin/streptomycin (1000 U/mL) (Invitrogen, Grand Island, NY, USA), 1% MEM amino acids solution (Invitrogen), 1% L-glutamine (Invitrogen) and 0.1% gentamycin (Invitrogen). Cells (2×105) were cultured in a 24-well plate (Corning, New York, NY, USA) with capillaries with or without MTA, for 24 hours for cell viability assay. Another culture was performed, including 2-capillaries/well with or without MTA, for 24 hours. In order to observe osteoclast differentiation, 100ng.mL−1 of recombinant (r) RANKL (Peprotech, Rock Hill, NJ, USA) was added to this culture, and every three days, half of medium and rRANKL (Peprotech) were changed, and the cultures were harvested on days 5, 6 and 8. RNA extractions were performed after 48h of cell incubation. The evaluation of the effect of aluminum and calcium on osteoclastogenesis was performed through RAW cells incubation with or without 40μg.mL−1 of aluminum oxide, 99.99% (Sigma) and calcium oxide Reagent Plus, 99.99% (Sigma), with/without rRANKL for 7 days.
Alpha mem
Manufactured by Merck Group
Sourced in United States, United Kingdom
Alpha-MEM is a powdered cell culture medium formulated for the in vitro cultivation of mammalian cells. It provides a balanced salt solution with amino acids, vitamins, and other nutrients required for cell growth and maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Dexamethasone (dex), by Merck Group (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Streptomycin, by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Ascorbic acid, by Merck Group (4 mentions)
Alpha mem, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
β glycerol phosphate, by Merck Group (4 mentions)
Saos 2, by American Type Culture Collection (3 mentions)
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
β glycerophosphate, by Merck Group (3 mentions)
L glutamine, by Merck Group (2 mentions)
Rabbit anti actin, by Merck Group (2 mentions)
α mem, by Merck Group (2 mentions)
Rrankl, by Merck Group (2 mentions)
54 protocols using alpha mem
1
Osteoclast Differentiation Assay
2
Osteoblast Matrix Formation Assay
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A mouse calvarial pre-osteoblastic cell line, MC3T3-E1 (MC) subclone 4, was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and maintained in alpha-minimum essential medium (alpha-MEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2. The culture medium was replaced twice a week. MC cells were seeded onto 35-mm dishes at a density of 2 × 105 cells/dish. After 2 days, the medium was replaced with the matrix formation medium (alpha-MEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 50 μg/mL of ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA)), with or without β-aminopropionitrile (BAPN) (0–2 mM; Tokyo Chemical Ind., Co., Ltd, Tokyo, Japan), and cultured for 2 weeks. The cell-matrix layers were washed with ice cold phosphate buffered saline (PBS) three times and stored at -20°C. The matrices were processed for biochemical/histological analyses or used for cell culture substrates as outlined below.
3
Culturing Primary Sarcoma and Cell Lines
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Established MG-63, U2os and Ost cell lines were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Sigma), L-glutamine (4 mmol/L), 100 U/mL penicillin and 100 μg/mL streptomycin (all from Invitrogen) at 37°C with 5% CO2. For primary sarcoma cells, specimens were obtained in accordance with Research Ethics Board approval from the Italian National Institute of Cancer G. Pascale and classified according to World Health Organization guidelines. Primary GCTB cell line derived from patients were freshly isolated as previously described [19 (link), 20 (link)] and grown in alpha-MEM (Sigma) supplemented with 10% FBS plus antibiotics. The experiments were performed in alpha-MEM plus 1% WF or 2% FBS (low serum) or 1% Benign effusion.
4
Osteoblast-Bone Marrow Co-culture
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Primary osteoblast isolated from DicergtRosaCreERT2 mice were seeded as 8,000 cells per well in 96-well plates and treated with 1μM 4-OHT for 3 days as described above. Bone marrow cells from wild-type mice were isolated and 200,000 cells per well were added on top of primary osteoblasts cultivated in alpha-MEM supplemented with 10 nM 1,25-dihydroxyvitamin D3 (Sigma-Aldrich, St. Louis, USA). Medium was changed every 3 days. After 9 days, cells were fixed and stained for TRAP activity.
5
Osteoclast Differentiation and Activation
6
Cell Culture Conditions for Comparative Study
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Macrophages RAW 264.7 (TIB-71), pre-osteoblasts MC3T3-E1 subclone 4 (CRL-2593), and kidney MDCK cells (CCL-34) were purchased from the American Type Culture Collection (ATCC). Pre-osteoblast MC3T3-E1 were cultured in alpha-Minimum Essential Medium Eagle Medium (alpha-MEM, Sigma-Aldrich, St. Louis, MO, USA), and both macrophage and kidney cells were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA). All culture media were supplemented with 10% fetal bovine serum (FBS, BenchMark, Gemini Bio Products, Sacramento, CA, USA), 1% penicillin streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 1% L-glutamine (BenchMark Gemini Bio Products, Sacramento, CA, USA), and 1.5 g/L sodium bicarbonate (Sigma-Aldrich St. Louis, MO, USA), and incubated until confluence at 37 °C in a 5% CO2 atmosphere.
7
Osteotropic Breast Cancer Cell Line
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The parental human MDA-MB-231 (MDA-231) as representative of triple-negative breast cancer cells and human osteoblast-like cells Saos-2 were purchased from ATCC (Manassas, VA). The osteotropic MDA-231-BT1 was generated by repeated passages in vivo and validated for their ability to colonize bone and to cause osteolysis47 (link). Tissue culture medium (DMEM and alpha-MEM) was obtained Sigma-Aldrich (Dorset, UK). All primary antibodies were purchased from Cell Signalling Biotechnology (MA, USA) except rabbit anti-actin was obtained from Sigma-Aldrich (Dorset, UK). Mouse macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK) and receptor activator of NFκB ligand (RANKL) was a gift from Patrick Mollat (Galapagos SASU, France).
8
Culturing MG63 Osteosarcoma Cells
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9
In vitro Erythropoiesis and VCN Determination
10
Osteotropic Breast and Prostate Cancer Cells
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The endocannabinoid 2AG and MAGL inhibitor JZL184 were purchased from Tocris Biosciences (Bristol, UK). The parental human breast MDA-MB-231, prostate PC3, osteosarcoma KHOS and Saos-2 were purchased from ATCC (Manassas, VA). The mouse osteosarcoma cells MOSJ were originally purchased from ATCC (Manassas, VA) and were a kind gift from Professor Dominique Heymann (INSERM, University of Nantes, France). The osteotropic human breast cancer cell line MDA-MB-231-BT (MDA-231-BT) was generated by Dr. Nadia Rucci (University of L'Aquila, Italy) and the osteotropic human prostate cancer cell line PC3-BT was a kind gift by Dr. Ning Wang (University of Sheffield, UK). Single cell clones of MDA-MB-231 were expanded and injected into the left ventricle of 4-week-old female BALB/c-nu/nu mice. A clone that generated bone-only lesions with significant osteolysis in 8-weeks was used [[34] (link), [35] , [36] (link), [37] (link)]. Tissue culture medium (D-Modified Eagle Medium (MEM) and alpha-MEM) was obtained from Sigma-Aldrich (Dorset, UK). All primary antibodies were purchased from Cell Signalling Biotechnology (MA, USA) except rabbit anti-actin, which was obtained from Sigma-Aldrich (Dorset, UK). Mouse macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Abingdon, UK) and receptor activator of NFκB ligand (RANKL) was a gift from Patrick Mollat (Galapagos SASU, France).
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