Comparison of Free Zinc Levels Determined by Fluorescent Probes in THP1 Cells Using Microplate Reader and Flow Cytometer

Free zinc is involved in signal transduction within mammalian cells, acting as a second messenger. Gold standard for its analysis is currently the use of metal-responsive fluorescent probes. The present study elucidates the impact of instrumentation used for measuring the resulting fluorescence. The free zinc concentration of THP-1 cells loaded with the fluorescent probes Zinpyr-1 (ZP1) or Fluozin-3 AM (FZ3) was determined using a microplate reader (MPR) and a flow cytometer (FC). Depending on the instrumentation, either low nanomolar (MPR) or picomolar (FC) concentrations of free zinc were observed. The concentrations measured from identical samples by MPR were about 40 (ZP1) or 165 (FZ3) times higher compared with FC. These results demonstrate that the choice of instrumentation has a fundamental impact on the determination of intracellular free zinc concentrations by low molecular weight fluorescent probes.