In flow cytometry it seems the biggest source of confusion and angst is compensation. As a first post, I’m going to re-publish a post I did elsewhere.
Compensation is somewhat tricky if you don’t understand the process. There are lots of good online tutorials and posts about compensation, but there are a few specific items I’d like to address and have “published” for viewing so I’ll throw my hat in the ring as well.
All fluorochromes will emit light across a range of wavelenghts, and if you are trying to take measurements for another fluor in a band that is included in this range, you will get background light (“spillover” or “spectral overlap”) from that other fluorochrome. To correct for this, most cytometry systems have some mechanism for “compensation” to remove this background. In essence, for any other parameter/detector that you are measuring, you want your single positive population to have the same Mean Fluorescence Intensity (MFI) as the negative population so that any positive signal in the other channels can be attributed to that particular color, and is not spillover from something else. In the case shown, FITC signal is being detected in the PE channel, and once compensated has the same mean fluorescence in the PE channel as the unstained.
Many companies now offer antibody-capture bead kits for compensation (Invitrogen, BD, Bangs Labs, Spherotech) and they usually include a “binding bead” and “non binding bead” in the same box. For example, BD offers “Compbeads” which bind to a specific species’ IgG (mouse, rat, hamster), so for a mouse-anti-Human CD4 antibody, you’d need the mouse binding bead, but for a rat-anti-mouse CD19 you’d need the rat binding bead. For Invitrogen’s ARC beads which are used to compensate their amine-reactive Live/Dead fixable stains, the binding beads are coated with amine groups so the reactive dye will bind to the bead. The Live/Dead dye will NOT bind to antibody-capture beads.
In order to correctly do this calculation, it is critical that you are comparing particles of the same type– if you are using stained cells, you want to compare the signals to the *same type* of unstained cells. If you are using beads, you want to compare to the exact same unstained bead. The “non-binding” bead is included in the kits for this purpose-they are the same bead as the binding one in the kit, but will not stain with the reagent and thus remain negative. You can include them both in the same tube, although you should wash the beads after staining to remove free dye if you do so.
Mismatches in your positive and negative particles will result in compensation errors, which could dramatically impact your data. More on that later… Mike