Time-lapse microscopy

Abstract: The directed migration of cells (chemotaxis) occurs not only during wound healing and inflammatory responses but also during embryonic development. However, the intracellular signaling pathways that enable a cell to detect a chemoattractant and subsequently migrate toward the source are not clearly defined. The Dunn chemotaxis chamber in conjunction with time-lapse microscopy is a powerful tool that enables the user to observe directly the morphological response of cells to a chemoattractant in real time. Here, we describe using the Dunn chemotaxis chamber to study the response of murine bone marrow-derived macrophages to colony stimulating factor-1. This is a particularly useful protocol as it can be adapted to study bone marrow-derived macrophages isolated from genetically modified mice and thus study the requirement for a specific protein in cell migration and chemotaxis.

Abstract: We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.

Abstract: The directed migration of cells (chemotaxis) occurs not only during wound healing and inflammatory responses but also during embryonic development. However, the intracellular signaling pathways that enable a cell to detect a chemoattractant and subsequently migrate toward the source are not clearly defined. The Dunn chemotaxis chamber in conjunction with time-lapse microscopy is a powerful tool that enables the user to observe directly the morphological response of cells to a chemoattractant in real time. Here, we describe using the Dunn chemotaxis chamber to study the response of murine bone marrow-derived macrophages to colony stimulating factor-1. This is a particularly useful protocol as it can be adapted to study bone marrow-derived macrophages isolated from genetically modified mice and thus study the requirement for a specific protein in cell migration and chemotaxis.

Abstract: Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion (1, 2). During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues (3) provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment. Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration (2). Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration. Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function (4), hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO(2) supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.