Mobility quantification of single cells, plasma membranes or cellular processes in dense cell cultures is a challenge, because tracking of individual cells is not readily possible. We used the recently developed software “SynoQuant” (designed by AWH; accessible on www.synosoft.de) to analyze the speed of randomly moving cultured cells, the dynamics of plasma membrane contraction and organelle trafficking. Primary cultures of brain pericytes, hippocampal neurons and chicken telencephalon cells were produced from Sprague Dawley rats[1], Wistar rats[2] and chicken embryos[3], respectively. All experiments were carried out in accordance with the guidelines of laboratory animal care in Kuwait University and the protocols were approved by Animal Resource Center. In some cases, cells were exposed to oxygen glucose deprivation (OGD) as described earlier[1] or maintained in the presence of drugs that affect cell mobility. Images were collected at various intervals over 15-48h in a temperature-controlled chamber on the phase-contrast microscope. Speed quantifications were obtained by segmenting cellular components and determining their individual velocities separately. Algorithms that use image subtraction (DiffMove’), or image correlation analysis (‘COPRAMove’) were used to analyse the motility speed of: 1. Intracellular vesicles in chicken telencephalon glia cells (CTG), 2. Somata and neurites in hippocampal neurons and 3. plasma membrane dynamics in pericytes. Differences between the groups were tested with Student’s t-test or with Wilcoxon-Mann-Whitney U-test. The speed of vesicles in CTGs was measured with both algorithms (Fig.1); the velocity of the organelles was slowed down by addition of staurosporine, a drug that reduced vesicle movement in the frog neuromuscular junctions[4]. Figs. 1B and C visualize vesicle traffic in an overlay image that was composed of two consecutive images, assigned to different color channels. Neurites and somata of hippocampal neurons were measured separately and their motility velocities were compared to the unprocessed series. The visual impression that the neurites moved slower than the somata, was confirmed by the ‘COPRAMove’ quantitative analysis (Fig. 2A, B). The motility of pericytes was examined to quantify membrane ruffling, cell constriction and pseudopodia extrusion. Figs. 2C-F show that the motility of pericytes was consistently and significantly (p < 0.01) decreased after 1-hour of OGD. Pericytes showed alternating periods of extension and retraction of pseudopodia under control conditions. In conclusion, this study has revealed that our new software could be used successfully to reconstitute and analyze cellular and subcellular movements in cell cultures.