Cells were plated at 2.5×104 cells per well in 12 well plates and cultured in maintenance medium for two days before changing to mineralization medium (maintenance medium+50 µg/ml ascorbic acid and 5 mM β-glycerol phosphate (βGP; Sigma) or 3 mM P-Cho (Sigma) as phosphate donors. PHOSPHO1 and TNAP inhibitors, MLS-0263839 and MLS-0038949 (both 30 µM) were added where indicated. This concentration was derived from our previous dose response experiments and showed maximum enzyme inhibition and no cellular toxicity [22] (link). Medium was changed every 3–4 days for up to 21 days. Cells were fixed for alizarin red staining or lysed for protein or mRNA extraction.
P cho
Manufactured by Merck Group
Sourced in Canada
P-Cho is a lab equipment product manufactured by Merck Group. It is used for the measurement and analysis of cholesterol levels. The core function of P-Cho is to provide accurate and reliable cholesterol testing capabilities in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using p cho
1
Mineralization Induction in Bone Cells
2
Mineralization Assay with Phosphate Donors
3
Purification and Characterization of Phospholipids
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CTP was purchased from Thermo Fisher Scientific (Mississauga, ON, Canada). Chelex 100 sodium form (50–100 mesh), AEP, P-Cho, and CDP-Cho were purchased from Sigma-Aldrich (Oakville, ON, Canada). P-Cho was treated with Chelex 100 resin as previously described prior to use in order to remove calcium, which inhibits the enzyme32 (link). All other chemicals were used without further purification. DNA sequencing was performed at The Centre for Applied Genomics at the Hospital for Sick Children (Toronto, ON, Canada). HR–ESI–MS was performed at the Alberta Glycomics Centre (Edmonton, AB, Canada). HPLC analysis was performed using a Prominence LC-20AT system equipped with a diode array detector (Shimadzu, Kyoto, Japan). FPLC purifications were performed using an NGC™ Chromatography System (Bio-Rad, Mississauga, ON). NMR spectra were recorded on an Agilent DD2 operating at 400 MHz for 1H and 100 MHz for 13C. A Varian Unity Inova operating at 121 MHz was used to record 31P NMR spectra.
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