35s sulfate

HUVEC cultures of the desired density were metabolically labeled with 0.1 mCi/ml ³⁵S-sulfate (Hartmann Analytic) in RPMI-1640 sulfate free medium (GIBCO Invitrogen) added 5 mM L-glutamine (Sigma) and with FCS reduced from 7 to 2% to increase labeling efficiency. After labeling for 24 hours, the culture medium was collected. The cells were washed in PBS and harvested in either lysis buffer (4.0 M guanidine-HCl, 0.1 M acetate buffer pH 6.5, 2% Triton X-100) or RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% Na-deoxycholate, 10 mM EDTA, 10 mM Na₄P₂O₇ and phosphatase inhibitor tablet freshly added). In order to remove unincorporated ³⁵S-sulfate, samples were subjected to Sephadex G50 fine (GE Healthcare) gel chromatography in buffer (0.05 M Tris-HCl, 0.05 M NaCl, pH 8). The ³⁵S -macromolecules were eluted in the void volume, while smaller molecules remained associated with the column. The amount of ³⁵S-sulfate incorporated in newly synthesized ³⁵S -macromolecules was determined by scintillation counting in triplicates. ³⁵S-macromolecules in HUVEC are almost exclusively comprised of PGs [16 (link)]. Protein content of cell fractions was determined in RIPA-lysates prior to G50 fine gel chromatography or in guanidine-lysates after changing the buffer on the G50 fine column.

Human myoblasts or myotubes were incubated in 100 μCi/mL (³⁵S)sulfate (Hartmann Analytic GmbH, Braunschweig, Germany) in standard medium for 24 h. Conditioned media were collected and centrifuged to remove cell debris and the cell fractions were lysed in Hepes buffer (pH 7.4) with 1% Triton X-100. To remove unincorporated (³⁵S)sulfate and to recover (³⁵S)sulfated macromolecules, medium and cell lysates were subjected to Sephadex™ G-50 Fine (GE Healthcare, Little Chalfont, UK) gel chromatography (Meen et al. 2011 (link)). The sulfated macromolecules were eluted in the void volume with 0.05 mol/L Tris–HCl, 0.05 mol/L NaCl, pH 8. The amount of (³⁵S)macromolecules isolated was quantified by scintillation counting. Samples were concentrated using Amicon Ultra centrifugal tubes with a molecular mass cut off at 3 kDa and the protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific).