Compensation Theory and Practical Implementation
- Compensation control requirementsfor the proper instrument setup:
- Unstained control sample should be the same origin and prepared with the same protocol as the cells used in the experiment;
- Single-color controls:
- Need to have single-color controls for all the fluorochromes used in the experiment;
- All controls should be treated in the same way as samples (such as: incubation, fixation, permeabilization);
- 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.
- Within each single-color control tube the unstained and positive particles have to be of the same origin (either cells or beads);
- Level of fluorescence in the controls should be same or brighter than in the samples.
- 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. For example, see below the "dedicated" channelsfor the most common fluorochromes:
- FL1: Ax488, FITC, EGFP, YFP;
- FL2: PE;
- FL3: PerCP-Cy5.5, PI, 7AAD;
- FL4: Ax647, APC, ToPro-3.
- Implementation - while monitoring 2-color dot plots, adjust compensation settings so that positively stained particle population (cells or beads) is directly in line with the unstained background particle population (cells or beads) and parallel with the "dedicated" channel axis.