To further understand fluorescent compensation, there is a tuturial by Mario Roederer at the following link: "http://www.drmr.com/compensation/index.html"
For surface staining of cells; usually three controls are needed for each experiment. The controls must undergo the same treatment (i.e., preparation) as all the tubes in an experiment. An unstained control is used to detect auto-fluorescence or background staining innate to the cells of interest. Auto-fluorescence can be a significant problem, particularly in systems that contain monocytes/macrophages, cultured cells, or activated cells. An isotype control (i.e., where an antibody is used that has the same immunoglobulin isotype as the test antibody, but a different specificity which is known to be irrelevant to the sample being analyzed) is needed to determine whether fluorescence emitted is due to non-specific binding of the fluorescent antibody. A positive control is highly desirable (although not always available) to prove that the test antibody and/or procedure is working properly. The positive control should include cells known to be positive for the marker of interest.
If more than one color (fluorescent conjugate) will be used within the same experiment, a single-color control must be prepared for each of the dyes used. This will allow for compensation from spectral overlap between the stains. An unstained control is also important for multi-color compensation. In any experiment where two or more fluorochromes (multi-color analysis) are used to characterize the cells of interest, the need for fluorescence compensation must be checked. Fluorescence compensation is the mathematical subtraction of the fluorescence of one fluorochrome from the fluorescence of another fluorochrome. This is necessary because emission spectra from multiple fluorochromes can overlap. For this reason, in a sample that contains 2 colors such as fluorescein isothyocyanate and phycoerythrin, fluorescence due to the FITC will be detected by the electronics that are set up to detect fluorescence produced by the phycoerythrin. If this were not subtracted from the phycoerythrin fluorescence, the phycoerythrin signal would be falsely elevated. To determine the correct amount of fluorescence compensation, cells stained with only one fluorochrome-conjugated antibody are analyzed individually. This must be done for each of the fluorochromes used in the experiment, with an unstained control as the initial region.
• MoFlo Homepage
• Flow Cytometry
• Cell Sorting
• Protocols and Sample Preparation
• Controls needed for Each Experiment
• What to Bring?
