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What is Flow Cytometry?
	Click to Enlarge Simplified diagram of a typical analytical flow cytometer Flow cytometry is a tool for measuring phenotypic, biochemical, and molecular properties of individual cells (or particles) using fluorescent probes which bind specifically to molecules associated with the cell. The cells are suspended in a fluid stream of PBS, and pass a laser which will excite the fluorescent probes (if the probe is excitable at the wavelength the laser is emitting). The light emitted from the probes is collected and processed by detectors specific for the wavelength emitted by the attached fluorochromes. The detectors translate the light into an electronic signal proportional to the intensity of the fluorescence emitted, represented as a pulse. In addition, information about the size and complexity (granularity) of the cell can be obtained when the cell passes through the laser beam. Flow cytometry can be further categorized into analysis and cell sorting.
Analysis
	Flow cytometry for the purpose of quantitative analysis of cellular properties is an invaluable research tool. The FACS Caliber allows up to 20,000 events per second to be analyzed, yet is sensitive enough to detect as few as 500 molecules of fluorescent probe per cell. The FACS Caliber is available to be used by individuals who have received training in its proper usage and receive a password through the facility.
Cell Sorting
	Click to Enlarge Diagrammatical representation of the process of cell sorting Cell sorting allows a desired population of cells (as assessed by analysis) to be sterilely collected into a collection tube of media. These cells may be cultured to enrich a population, used directly in an experiment such as RNA isolation, or be injected into an animal model. Cells which are selected to be collected are electronically charged in individual droplets, and then deflected into the appropriate container. A high degree of purity can be achieved, and multiple fluorochromes can be used for selection. The FACS Vantage SE can sort up to 25,000 events per second, but can only be used by a trained operator. The average rate of sorting is 20 million cells analyzed per hour.
Sorting Information
	Regular sorting: Regular sorting will process up to 4500 events per second. This is the gentler method which is best for fragile cells, cells that don't grow well, or cells that fiercely adhere to one another at the concentrations necessary to turbo sort. Cells should be concentrated at 5-10 million cells per ml. For more information about what to bring for regular sorting, see Tips for Successful Sorting. Turbo Sorting: Turbo sorting uses increased sample and sheath pressure, as well as increased drop drive frequency to allow 10,000-15,000 events per second without significant loss in yield, but can go up to 25,000 events per second if the loss in yield is not a factor. The increased stress on the cell due to the increased velocity makes fragile cells and cells which do not grow well poor candidates for turbo sorting. In addition, cells which cooagulate at higher concentrations are not recommended for turbo sorting because of the 15-20 million cells per ml requirement. For relatively hearty cells, turbo sorting is a great option. For more on what to bring for turbo sorting, see Tips for Successful Sorting. High-speed Enrich: This method is used to collect a large number of a small percentage of positive cells (less than 5% of the total) at high speed (25,000 cells per second) for a 5-10 fold enrichment of the population. The sorter will collect all of the desired cells, but will also collect undesirable cells in the drop envelope with the desired cell. The new, enriched population may then be re-sorted to increase the purity using standard sorting, or used at 50% purity. The advantage of enriching a population and re-sorting it is that it will take less time overall, thus decreasing the cost. A 10 hour sort can be reduced to 1-2 hours! This is best if you have plenty of starting material that is hearty enough to be sorted twice. A major disadvantage is the significant loss in yield (up to 50%), which can be countered by doubling the amount of starting material. The cells should be prepared as for a high-speed sort. Accudrop: This is not a type of sorting, but a vital part of our machine. It is a video monitor which shows the core stream as well as the side streams. Accudrop allows the operator to set the drop delay more accurately, as well as see that the side streams are uniform. During sorting, the operator can see that the desired cells are going left or right because of the fluorescence of the sorted cells. 96-well plates: Typically, a population is selected and one cell per well is deposited into media of your choice. You will need to prepare the plates before coming to sort. Any number of cells can be deposited in each well. Much easier than dilutional pipetting!
Literature Available in the FACS Laboratory
	Cytometric Bead Array Proliferation and Cytokine Expression Mouse Intracellular Cytokine Cell Cycle BD LSR Flow Cytometer Calcium Fux Rat FC Block BD FACS Calibur Peptide Mix Protocol BD Cellquest Pro BRDU Flow Kit Overview Human cell Staining Intracellular Flow Cytokine Analyzing Data on Cellquest Acquiring Data on Cellquest