Enabling techniques for separating target cells, such as subgroups of white blood cells and cancercells from blood and other body fluids, are necessary for lab work facilitation, inline integration withhigh-end bioanalytical instrumentation, and for point-of-care or home testing. The work of this thesisaddresses this need by further studying an equilibrium-based ultrasonic wave-based technology calledisoacoustic focusing to characterize and gain access to cells. In this size-insensitive technique, cells aresuspended in a microchannel filled with an inhomogeneous medium where the interaction of diffusion,gravity, and acoustic radiation shapes a smooth gradient profile for the acoustic impedance of themedia orthogonal to the flow. While flowing, the acoustic radiation force pushes cells towards theirisoacoustic point where the acoustic contrast and radiation force become zero and cells’ acousticallyinduced sideways displacement ceases. The first two included papers in this thesis uncover in detail theacoustophoretic motion of cells suspended in homogeneous and inhomogeneous media in a stop-flowcondition. Cell three-dimensional trajectories were measured by a defocused-image tracking approach,and the technique’s applicability for tracking cells was assessed by determining the associated errorwhen measuring the out-of-image-plane component of the tracks. In a homogeneous medium, for cellswith near-zero acoustic contrast, strong effects of buoyancy and acoustic streaming were observed,and small distributions of cell properties within a population resulted in large differences in thecell motion patterns. The second article shows how cells migrate towards their iso-acoustic point inacoustic impedance gradient media while acoustic streaming is substantially suppressed. This enabledthe readout of the effective acoustic impedance of neutrophils and K562 cancer cells. A numericalmodel was introduced to estimate the acoustic energy density in the acoustic impedance gradientmedia by tracking particles of known properties. To examine cell separation based on the knowledgeacquired in the two first studies, the third paper presents the use of gradient acoustic focusing anddense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from wholeblood in a label-free and flow-through format. By modifying the medium and thus tuning the contrastfactor of the cells, PBMCs were enriched relative to RBCs by a factor of 3600 to 11000 and with aseparation efficiency of 85%. Such a level of RBC depletion is high compared to most other microfluidicmethods and similar to density centrifugation. In the fourth Paper, we show that cell compressibilitycan be determined at the isoacoustic point by an independent measurement of the density of each cell.Cells were pre-sorted off-chip in linear, continuous, and reproducible density gradients, and fractionswith known densities were fed into an isoacoustic focusing device. The relation between density andcompressibility for two cell types was investigated, and it was found that for increasing density ofK562 cells, the compressibility decreases. For neutrophils, the compressibility was measured, and aslight change was observed with increasing density.