Free flow electrophoresis of cell suspensions in buffers containing sodium chloride was investigated using a modified procedure and the new apparatus Octopus PZE. The major methodical innovations are upward fluid flow, margin buffers flowing through the electrophoresis chamber at both sides of a central cell suspension buffer, adjacent to the electrode membranes, and a sample injection device which focuses the cells hydrodynamically to the middle of the chamber thickness. Mononuclear leukocytes, suspended in a buffer containing 35 mM NaCl, could be fractionated with the same accuracy as by conventional free flow electrophoresis, operated with a single NaCl-free chamber buffer. However, testing the vitality of separated cells with the help of the calcium indicator FURA2-AM clearly demonstrated the biological importance of the presence of a minimum amount of sodium chloride during cell electrophoresis. Only if at least 35 mM NaCl were present could an undisturbed cytosolic Ca2+ metabolism be maintained for the time of a free flow electrophoresis cell separation experiment.