INTRACELLULAR NA+ MEASUREMENTS USING SODIUM GREEN TETRAACETATE WITH FLOW-CYTOMETRY

Intracellular sodium concentration ([Na+](i)) can be measured using SBFI with image analysis or spectrofluorometry. However, SBFI cannot be used with most now cytometry systems, due to its requirement for dual excitation in the UV range. Recently a new sodium indicator, Sodium Green, was developed which can be used with now cytometry to measure [Na+](i). Adequate staining of cells with Sodium Green is dependent upon both dye and cell concentration, time and temperature of loading, use of the non-ionic detergent Pluronic, and the cell Line used. Both Sodium Green and SBFI give similar calibration curves using the ionophore gramicidin, Sodium Green has a small pH dependence, but this should not affect most applications, Since a fluorescence ratio technique cannot be used with Sodium Green, we used a ratio of dye fluorescence to forward angle Light scatter to normalize for cell size variations in the population, The basal [Na+](i) obtained using the Sodium Green technique in CHO IS1 cells was 19 mM, in reasonable agreement with previous studies using SBFI in other cells. (C) 1995 Wiley-Liss, Inc.