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Found 3 matching solutions for this experiment
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The human osteoblasts were cultivated in modified Eagle’s osteogenic cell culture medium (MEM; Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% amphotericin B, and 1% HEPES buffer (all from Gibco-Invitrogen, Darmstadt, Germany) without calcium, and the osteogenic additives dexamethasone (100 mM), L-ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany).The osteogenic differentiation of the human primary osteoblasts was confirmed by immunohistochemical detection of the enzyme alkaline phosphatase using a fuchsin+substrate chromogen (DAKO, Hamburg, Germany). For the further tests, the isolated cells were cultured in 25-cm2 flasks with 8 ml of osteoblasts in the same medium but without the 1% penicillin/streptomycin and under standard cell culture conditions (5% CO2 and 37°C). |
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| The human osteoblasts were cultivated in modified Eagle’s osteogenic cell culture medium (MEM; Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% amphotericin B, and 1% HEPES buffer (all from Gibco-Invitrogen, Darmstadt, Germany) without calcium, and the osteogenic additives dexamethasone (100 mM), L-ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany).The osteogenic differentiation of the human primary osteoblasts was confirmed by immunohistochemical detection of the enzyme alkaline phosphatase using a fuchsin+substrate chromogen (DAKO, Hamburg, Germany). For the further tests, the isolated cells were cultured in 25-cm2 flasks with 8 ml of osteoblasts in the same medium but without the 1% penicillin/streptomycin and under standard cell culture conditions (5% CO2 and 37°C). |
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the trabecular bone was cut into small pieces and thoroughly washed with commercial standardized Joklik’s modified MEM serum-free medium, to remove no adherent marrow cells. The pieces were incubated with rotation at 37 °C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase, and collagenase digestion was stopped by the addition of Iscove’s modified medium containing 10% fetal bovine serum (FBS). Between eight and ten pieces from each patient were then placed in 25 cm2 flasks and cultured in IMDM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B until confluence; the culture medium was changed every 2–3 days. |
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| Protocol tips |
| the trabecular bone was cut into small pieces and thoroughly washed with commercial standardized Joklik’s modified MEM serum-free medium, to remove no adherent marrow cells. The pieces were incubated with rotation at 37 °C for 30 min with the same medium containing 0.5 mg/ml type IV collagenase, and collagenase digestion was stopped by the addition of Iscove’s modified medium containing 10% fetal bovine serum (FBS). Between eight and ten pieces from each patient were then placed in 25 cm2 flasks and cultured in IMDM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 U/ml mycostatin, and 0.25 µg/ml amphotericin B until confluence; the culture medium was changed every 2–3 days. |
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Three thousand normal human osteoblasts were seeded in 24-well plates (Fisher Scientific) containing 1 mL media with different MgCl2 concentrations 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM and 16.0 mM MgCl2 concentrations (Fisher Scientific). Each condition was triplicated and growth medium was changed every three days. The culture plates were incubated under 37̊C and 5% CO2 for following times: 16 hours, 7 days, 10 days, 14 days and 21 days. Cell attachment was quantified at 16 hours, through direct cell counts and normalized to the cell seeding density. Cell proliferation was determined at time points of 7, 10, 14 and 21 days. |
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| Three thousand normal human osteoblasts were seeded in 24-well plates (Fisher Scientific) containing 1 mL media with different MgCl2 concentrations 0 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM, 8.0 mM and 16.0 mM MgCl2 concentrations (Fisher Scientific). Each condition was triplicated and growth medium was changed every three days. The culture plates were incubated under 37̊C and 5% CO2 for following times: 16 hours, 7 days, 10 days, 14 days and 21 days. Cell attachment was quantified at 16 hours, through direct cell counts and normalized to the cell seeding density. Cell proliferation was determined at time points of 7, 10, 14 and 21 days. |
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