Introduction to Turku BioImaging key technologies (see the Turku BioImaging Black Book for more information).
Flow cytometry is a method for analyzing cell populations. The technique allows thousands of cells to be evaluated. Most fluorophores available for fluorescence microscopy can be used in flow cytometry. The most common applications are phenotyping of cells, the counting of cells transfected with fluorescent proteins, and cell cycle analysis. Cells in suspension are injected into the instrument, where they are accelerated and hydrodynamically focused into a straight line of flow. The flow crosses a laser beam, and every cell is individually illuminated. Fluorophores attached to or inside a cell are excited and the emitted light is gathered by collection optics. The cells to be analyzed must be prepared as a single cell suspension. Many samples, including peripheral blood mononuclear cells (PBMCs), confluent cell lines, or bacteria, can be injected directly. Adherent cells must be detached. Tissue must be homogenized before analysis. Practically all commercial fluorophores can be measured with flow cytometry. Turku BioImaging offers several instruments from BD for flow cytometry.
Flow cytometric cell sorting is a method for enrichment of cell populations, affording very high purity. In addition to regular analysis, between one and four cell subpopulations can be selected and sorted into tubes. These cells can be re-cultured, or used for PCR or as a source of microarray data. As in basic flow cytometry, cells are accelerated into the flow, excited with a laser beam and the emitted light analyzed. After laser excitation, the flow is broken into drops. Each drop contains only one cell. If the cell inside the drop meets sorting criteria, the drop is electrically charged. The drop goes through deflection plates with a high potential, and charged drops are diverted from the main stream into a collection tube. In this manner, every cell is analyzed and a decision on sorting is made for each individual cell. This means that the sorted cells are of very high purity. The technique can be used for instance to enrich GFP-positive cells or to sort antibody-labeled cells. Cell sorting is performed as a service by Turku BioImaging.
Flow cytometry data analysis
The analysis of flow cytometry data is often an even bigger task than the sample preparation and measurement. Turku BioImaging offers Flowing Software, a free tool for analysing the datasets created by flow cytometers. Flowing Software is able to perform all basic tasks, such as visualizing the data with dot plots and histograms, gating and calculating statistics. The data copied from the instruments can be opened and analyzed in a very similar manner to commercial software packages. The true power of the software becomes clear when the number of data files increases. Flowing Software offers unique tools for easy automation of analysis paths and the collection of results. Everything is based on objects and arrows connecting them. By connecting objects the user is able to create an analysis path, which performs all the tasks needed, from visualization to data collection. The automation does not require any special computational skills, because everything is done via the clear graphical user interface. Flowing Software can be downloaded from www.flowingsoftware.com.