Thymidine 5 monophosphate p nitrophenyl ester sodium salt

Cell layers were washed with PBS and harvested in a 10mM Tris-HCl, 2mM PMSF, 0.2% Igepal solution. Samples were frozen at −80°C until assayed, at which time they were thawed, homogenized on ice and centrifuged at 12,600 rpm for 10 min at 4°C. Supernatants were used to assay for TNAP and Enpp1 activities using substrates p-nitrophenyl phosphate (Sigma Aldrich) and thymidine 5′ – monophosphate p-nitrophenyl ester sodium salt (Sigma Aldrich), respectively, and absorbance was measured at 405nm using a microplate reader. A standard curve was used to correlate absorbance units to enzyme concentration, and values were normalized to total protein content (obtained from BCA assays (Pierce) performed with cell lysate supernatants).

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) activity was determined following homogenization of hydrogels in RIPA. The ENPP1 substrate, thymidine 5’-monophosphate p-nitrophenyl ester sodium salt (Sigma), was dissolved at 1 mg/mL in diethanolamine buffer, and relative ENPP1 activity was measured via absorbance at 405 nm and normalized to hydrogel DNA content using PicoGreen (Invitrogen). For measurement of pyrophosphate (PP_i) concentration, hydrogels were transferred to new wells with 500 μL inductive medium and cultured for 24 hours. 40 μL of medium or standard (sodium pyrophosphate tetrabasic, Sigma) was then combined with 10 μL each of PPiLight^™ converting and detection reagent (Lonza, MD, USA) in 96 well plates and luminescence was read once every minute for 10 minutes. The slope of luminescence versus time was used to determine pyrophosphate concentrations.