Tri carb 1900 tr scintillation counter

Palmitic acid uptake was evaluated according to the Chavez and Summers procedure [88 (link)]. Briefly, L6 myotubes were starved for 3 h before palmitic acid uptake assessment. Afterwards, myotubes were incubated for 20 min with either Krebs-Ringer buffer (KRB; 140 mM NaCl, 20 mM HEPES, 5 mM KCl, 2.5 mM MgSO₄, 1 mM CaCl₂) or KRB buffer supplemented with 100 nM insulin (Gensulin, Bioton, Poland). Subsequently, the cells were exposed to medium containing 2 mM palmitate complexed with 1% bovine serum albumin (Sigma Aldrich), with the addition of 9,10-[³H(N)]-palmitic acid (1 µCi/mL). After medium removal, the reaction was terminated with ice-cold PBS buffer and the cells were solubilized in 0.05 N NaOH. The resulting fluid was transferred to 5 mL scintillation vials and counted using a Packard TRI-CARB 1900 TR scintillation counter. Radioactivity was normalized concerning the protein concentration.

palmitic acid uptake was evaluated according to the procedure by Chavez and Summers (2003) (link). ADMSCs-derived adipocytes were starved in a serum-free medium for 3 h prior to experiment initiation. To start the uptake assay, a Krebs-Ringer HEPES buffer containing palmitic acid (Sigma-Aldrich, St. Louis, MO, United States) bound to bovine serum albumin (fatty acid–free, Sigma-Aldrich, St. Louis, MO, United States) and radiolabeled 9,10-³H palmitic acid (1 μCi/mL, Perkin Elmer, Shelton, CT, United States) was added to each well for 5 min at 37°C/5% CO₂. Then, the plates were placed on ice, and each well was intensively washed with ice-cold PBS buffer. The residual washing buffer was completely aspirated, and the cells were lysed in protein lysis buffer. Half of the lysates were used for liquid scintillation counting, and the rest were used for protein measurement. Radioactivity was measured using a Packard TRI-CARB 1900 TR scintillation counter and normalized to protein concentrations.