Simultaneous HPLC determination of soluble signaling molecules and metabolic status in neuroblastoma cell cultures

Several laboratories have explored the capacity of the anion exchange chromatography method in evaluating distinct forms of inositol phosphates in a single analytical process. We describe a straightforward HPLC method to analyze simultaneously inositol phosphates and nucleotides present in a neuronal cells culture. The method was applied to neuroblastoma cells grown in standard media. The culture has an optimal metabolic state between 45% and 95% of confluence, but there was a rapid metabolic deterioration when the culture density exceeded 100%, which is important to consider when cell stimulation studies are carried out in culture. This method also allows the quantification since 0.5 to 50 nanomoles of nucleotides present in a single confluent culture from a T-75 flask containing 8 million cells. In addition, cyclic adenosine monophosphate (cAMP) and adenosine monophosphate (AMP) were eluted without overlapping. Therefore, the method has proven to have sufficient sensitivity to determine quantitative changes in nucleotides and inositol phosphates in a sample with low cell density. Moreover, the simultaneous determination of signaling and metabolic molecules allows obtaining a rapid and suitable control of the metabolic status in studies on cell stimulation that should be applicable to other types of cultured cells.