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Compensation Theory

  1. Compensation control requirements for the proper instrument setup:
    1. Unstained control sample should be the same origin and prepared with the same protocol as the cells used in the experiment;
    2. Single-color controls:
      1. Need to have single-color controls for all the fluorochromes used in the experiment;
      2. All controls should be treated in the same way as samples (such as: incubation, fixation, permeabilization);
      3. Should contain particles (either cells or compensation beads) stained with each fluorescence marker individually;Note: Most common fluorescence markers: fluorochrome-labeled antibody (directly conjugated or via biotin-SA-fluorochrome system), fluorescent proteins, functional probes, etc.
      4. Within each single-color control tube the unstained and positive particles have to be of the same origin (either cells or beads);
      5. Level of fluorescence in the controls should be same or brighter than in the samples.
  2. Compensation goals — unstained and stained particles should show the same mean value (or median for digital instruments) in all the “bystander” channels except the one “dedicated” for it.