Fluorometric hexokinase glucose assay

Primary hepatocytes were washed with PBS and incubated in a glucose-free DMEM starvation medium (1% HEPES, 2% BSA, 44 mM NaHCO₃, 2.5% glutamine and 1% penicillin-streptomycin) for 8 h. Hepatocytes were provided with a supplemented glucose production DMEM medium (without phenol red, 2 mM sodium pyruvate and 20 mM sodium L-lactate as gluconeogenic substrates) with indicated stimulants and glucose was measured by fluorometric hexokinase glucose assay (abcam, Cambridge, UK) according to the manufacturer’s instructions over 8 h. Assay results were normalized for protein content using a BCA protein assay kit (Sigma-Aldrich, Steinheim, Germany).

Primary hepatocytes were washed with PBS and incubated in a supplemented glucose-free DMEM starvation medium (1% HEPES, 2% BSA, 44 mM NaHCO₃, 2.5% glutamine and 1% penicillin-streptomycin) for 8 h. Hepatocytes were then provided with glucose production supplemented DMEM medium (without phenol red, 2 mM sodium pyruvate and 20 mM sodium L-lactate as gluconeogenic substrates) and with the indicated stimulant. Then the glucose was measured by fluorometric hexokinase glucose assay (abcam) according to the manufacturer’s instruction over 8 h. Assay results were normalized for protein content using a BCA protein assay kit (Sigma-Aldrich).