Compensation Theory
- 1. Compensation control requirements for the proper instrument setup:
- a. Unstained control sample should be the same origin and prepared with the same protocol as the cells used in the experiment;
- b. Single-color controls:
- i. Need to have single-color controls for all the fluorochromes used in the experiment;
- ii. All controls should be treated in the same way as samples (such as: incubation, fixation, permeabilization);
- iii. 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.
- iv. Within each single-color control tube the unstained and positive particles have to be of the same origin (either cells or beads);
- v. 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.