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Four steps must be taken to assure proper compensation. These steps should be taken with every experiment where the cell types differ, where the reagents differ, or where any instrument settings change--daily compensation is thus probably not frequent enough!
(1) The compensation tube must consist of cells that are unstained as well as cells that are singly-stained with the fluorescent probes. The stained (positive) cells must have the same autofluorescence (when they are unstained) as do the unstained (negative) cells in the compensation tube (e.g., all are lymphocytes).
(2) The PMT voltages must be set high enough guarantee that the negative population is off the axis in every channel.
(3) An analysis gate is set so that only cells with identical autofluorescence characteristics are viewed (e.g., a lymphocyte gate). An analysis gate is also set to include all of the negative cells and all of the positive cells.
(4) The centers of the positive and negative cell populations are aligned by matching the median fluorescences.