3h riboflavin

RF uptake assay was performed with confluent monolayers of H9C2 cells by incubating with KR buffer containing [ 3 H]-riboflavin (6.2 Ci/mmol with 96% radiochemical purity was purchased from Moravek Biochemicals, Inc, USA) at 37 °C for 10 min. Labeled and unlabeled RF were added to the incubation buffer at the onset of uptake experiment and the reaction was terminated after 10 min of incubation by the addition of 2 ml of ice-cold KR buffer followed by immediate aspiration. Cells were then digested by incubating at 80˚C for 20 min with 1N NaOH followed by neutralization with HCl. Digested cell contents were harvested and then counted for radioactivity using a Scintillation counter (Beckman Coulter LS6500). The protein content of cell digests was measured from the experimental and control wells using Bradford assay and used for the normalization of uptake data. To determine the effect of pH on RF uptake, the assay was performed with KR buffer adjusted to different pH ranging from 5.5, 6.5, 7.5 and 8.5. Na + dependency of RF uptake was tested by isosmotically replacing the Na + in the incubation buffer with other monovalent cation, namely lithium or with the nonionic mannitol.