Multiwell 14CO2-capture assay for evaluation of substrate oxidation rates of cells in culture

Abstract

14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially available culture and filter plates designed for use in a multiplate scintillation spectrophotometer enabled substrate oxidation rates to be evaluated for cells in a 24-well plate format without the need to dislodge the cells from the culture substrate as is required in traditional methods. Evaluation of radioactivity captured in potassium hydroxide-saturated filters was accomplished by adding scintillation fluid to the filter plate wells and counting. Alternatively, filters could be removed and placed in vials for evaluation in a conventional scintillation counter. This method was applied to the oxidation of 14C-glutamine by human breast cell lines and demonstrated concentration-dependent linear accumulation of captured counts.