(Go back one page to Requirements for Proper Compensation, or back to the introduction)

Correct compensation requires the ability to compare a population of cells stained with only a single color against an unstained population of cells. In general, this requires n tubes to be set up for compensation, one for each color, with unstained cells mixed in to the tube after the cells have been stained and washed.

Alternatively, one could take all of the singly-stained cell samples, and mix them into a single compensation tube. Now you would end up with n+1 different populations (singly-stained plus one unstained population) for compensation. Each required compensation would be performed in turn, by focussing only on the relevantly stained cells and unstained cells.

Another alternative is to stain a single population of cells with antibodies that would bind to exclusive populations of cells (for instance, in three-color analysis of peripheral blood lymphocytes, one could use CD19, CD8, and CD4--ignoring the CD4+CD8+ cells, which are very rare).

These latter two alternatives result in adequate compensation, with the caveats that the singly-positive cells can be uniquely identified (gated on), and that the autofluorescence of these populations is all the same. In using PBL samples, these two criteria are generally met.

However, when performing higher-order compensation (for example, 4 or more colors), then it becomes highly desirable, if not necessary, to use individual compensation samples. At a minimum, this is because it can become impossible to gate uniquely on singly-stained populations when so many dimensions are involved. But also, it becomes difficult to select this many unique and exclusively-expressed bright antigens.

An excellent approach (for compensating for PBL experiments) would be to have CD8 (a very bright antigen) available on every color. Separate tubes of cells are stained with each different CD8 conjugate and analyzed separately to set the compensations. CD8 is also a good choice, since there are typically many CD8-negative (or even CD8-dim) cells against which compensation is performed.

Go on to examples of 2-color compensation.