Surfaces were gamma irradiated prior to cell culture. MG63 cells were cultured as described above. Media were changed at confluence, and cells were harvested 24 hours after confluence, rinsed twice with 1xPBS, then stored at −20°C overnight for biological assays. Cell lysate was assayed for DNA content (P7589, Invitrogen) and total protein content (23225, Pierce). Alkaline phosphatase specific activity was measured as a function of p-nitrophenol production from p-nitrophenylphosphate at pH 10.2 and normalized to total protein. Media were assayed for osteocalcin (OCN, BT-480, Biomedical Technologies, Inc.), VEGF (DY293B, R&D Systems), OPG (DY805, R&D Systems), BMP2 (900-K255, PeproTech) and BMP4 (DY 314, R&D Systems). Data were normalized to total DNA content. Experiments were performed at least twice to ensure validity of the results.
P7589
Manufactured by Thermo Fisher Scientific
The P7589 is a laboratory instrument designed for conducting various analytical and experimental procedures. It is a versatile piece of equipment that can be utilized in a range of scientific applications. The core function of the P7589 is to provide a controlled and consistent environment for carrying out these processes. Further details about the specific intended use or capabilities of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
T7531, by Horiba (2 mentions)
Ab176723, by Abcam (2 mentions)
Dy805, by R&D Systems (1 mentions)
Dy293b, by R&D Systems (1 mentions)
S220 focused ultrasonicator, by Covaris (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
D4014, by Zymo Research (1 mentions)
Zirconia silica beads, by Thermo Fisher Scientific (1 mentions)
5 protocols using p7589
1
Osteogenic Differentiation Evaluation
2
Comprehensive Liver Metabolic Analysis
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Prior to analysis, frozen livers were powdered using a liquid nitrogen cooled mortar and pestle to obtain homogeneous liver samples. To measure liver DNA content (ng dsDNA/g tissue), 10–20 mg of powdered liver was sonicated in 200 μL DEPC H2O and dsDNA determined by fluorometric assay (Cat. # P7589, Invitrogen, Waltham, MA).
Total liver lipids were extracted from powdered liver samples. Briefly, 10–20 mg of sample was sonicated in 100μL PBS. 1 mL of 100% ethanol was added to each sample and vortexed for 10 minutes. Following 5 minutes of centrifugation at 16,000xg at 4°C, supernatant was transferred to a fresh tube for analysis of liver triglycerides (Cat. # T7531, Pointe Scientific Inc., Canton, MI).
Hepatic NADH and NAD were quantified by fluorometric assay (ab176723, Abcam, Cambridge, UK). Hepatic ATP concentrations were assessed as previously described (Geisler et al., 2016 (link)).
Total liver lipids were extracted from powdered liver samples. Briefly, 10–20 mg of sample was sonicated in 100μL PBS. 1 mL of 100% ethanol was added to each sample and vortexed for 10 minutes. Following 5 minutes of centrifugation at 16,000xg at 4°C, supernatant was transferred to a fresh tube for analysis of liver triglycerides (Cat. # T7531, Pointe Scientific Inc., Canton, MI).
Hepatic NADH and NAD were quantified by fluorometric assay (ab176723, Abcam, Cambridge, UK). Hepatic ATP concentrations were assessed as previously described (Geisler et al., 2016 (link)).
3
Comprehensive Liver Metabolic Analysis
4
Chromatin Shearing and Quality Assessment
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Sorted fixed cells were pelleted (800 rcf,10 minutes, 4°C). Cell pellets were resuspended in ChIP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 nM NaCl, 0.5% NP-40, 0.25% Sodium Deoxycholate, 0.1% SDS, 1x protease inhibitors (Sigma, 05056489001)) and incubated on ice for 10 min. Nuclei were collected by centrifugation (1000 rcf, 5 min, 4°C). The nuclei pellet was resuspended in shearing buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.25% SDS, 1x protease inhibitors) and then sheared to a size range of 200–500 bp on a Covaris S220 Focused-ultrasonicator (16 min, 2% Duty Cycle, Peak Power 105W, 200 cycles per burst, 6°C). Sheared chromatin was centrifuged (10,000 rcf, 10 min, 4°C) to remove insoluble material. The DNA concentration of the sheared chromatin was determined by fluorescent quantification (ThermoFisher, P7589). Shearing was assessed by agarose gel electrophoresis after DNA clean-up: chromatin was incubated for 30 min with RNase A, cross-links were reversed overnight at 65 C, and then DNA was column purified (Zymo Research, D4014).
5
Quantifying Residual DNA in Decellularized Lungs
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Decellularized lung tissue was quantified for residual double stranded DNA (dsDNA) using fluorescent nucleic acid staining (Thermo Fisher Scientific, P7589). Lung tissue slices were freeze dried and homogenized with 0.1 mm zirconia silica beads (Thermo Fisher Scientific, Waltham, MA, USA, cat.no. 3488) in a fast prep bead beater (MP fastprep96, Nordic Biolabs, Täby, Sweden). Samples were centrifuged at 6000× g for 3 min and supernatants were analyzed for dsDNA quantification according to manufacturer’s instructions.
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