X-ray measurement of intracellular chloride and other ions in mammalian cells

Intracellular chloride ion (Cl−) is known to have various roles in the cellular growth, modulation of cell cycles and volume, and in pathological disorders such as cancers, neurological and cardiovascular disorders. Various methods employed to measure intracellular Cl− requires lengthy procedure and measurement duration, bulky and advanced instruments, or incapable to measure low to single-cell sample quantity. In this study, we report the measurement of intracellular chloride and other ions in mammalian cells using the FROZEN freeze-drying method for preparing adherent cell cultures for analysis. The sample preparation does not require chemical fixation and enables direct measurement by X-Ray analytical methods such as X-Ray Fluorescence (XRF) or Energy Dispersive X-Ray Fluorescence (EDS). Cell staining showed that the cells retained their content and location after preparation, which enables single-cell level analysis. Treatment of Furosemide to the cells disrupted the Cl−, K+ and Na+ transport, causing changes in the intracellular ions. Additionally, the intracellular Cl− of the dried cells was successfully measured, as low as 0.9 mM. The XRF and EDS measurements showed that treated cells displayed a significant reduction of intracellular Cl− on all tested cell lines. However, no significant changes were detected in intracellular K+. Furthermore, EDS analysis on single cells showed a significant decrease in intracellular Na+ in most cell lines. These results demonstrate the efficiency and simplicity of the proposed method in intracellular ion analysis, allowing quick and simple preparation to measure biological samples with high sensitivity for various intracellular ions, which may be applicable in the diagnosis of diseases