As a general requirement, all samples should be prepared in a single-cell suspension form at a concentration of 6-8 million per ml to 10-20 million/ml (depending on cell type), after all labeling and filtration steps, or in a volume of 500 µl if only low cell numbers are available. DNA analysis requires fewer cells/mL. Immediately before it's brought for sorting, samples should be filtered through a 35-50 micron filter to remove any clumps or aggregates prior to analysis. This will ensure the removal of any larger clumps before the sample is introduced into the cell sorter. If this is not performed, and your sample does contain clumps, it can clog the flow cell, which will cause a considerable delay in the sorting of your sample as that clog would need to be removed, and the machine re-calibrated.
In order to correctly interpret flow cytometric data, there are several controls that need to be included in every experiment: unstained cells, and depending on the application, negative and/or positive controls. For multicolor analyses, compensation controls are needed to correct for the overlap in the spectral emissions of two or more fluorochromes. There needs to be one compensation control for each fluorochrome used in the experiment. The unstained control must be your cells, but the single-color controls can be cells or antibody-capture beads. There is an excellent link on the UCLA Flow Cytometry web site with procedures for numerous flow cytometry protocols. That link is: http://cyto.mednet.ucla.edu/. Leslie can also supply you with various protocols for flow cytometric analysis or sorting.
• MoFlo Homepage
• Flow Cytometry
• Cell Sorting
• Protocols and Sample Preparation
• Controls needed for Each Experiment
• What to Bring?
