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8-peak beads, sometimes called “rainbow” beads, are a set of beads in a single vial that contains 8 different populations that differ only in the amount of fluorophore contained within them. One of the peaks, termed Peak 1, is unlabeled, and the additional seven, termed Peaks 2-8, contain increasing amount of fluorophore. 8-peak beads are designed to fluoresce in all channels on most flow cytometers and cell sorters. These beads are used to check fluorescence sensitivity and resolution by measuring the position of the unlabeled peak and the separation between all of the peaks, respectively. They are also used to check linearity in fluorescence detection channels by correlating the amount of fluorophore on each population of bead with the position on the scale onto which the flow cytometer places the beads. You may have noticed that when you use your 8-peak beads that your peaks have different CVs and intensities – some are wider and taller than others. But do you know why? If not, how do you know if your cytometer or cell sorter is performing correctly? Here’s everything you need to know about using your 8-peak beads.

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FMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them. When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. It’s a strong negative control because the left out marker in the FMO control allows you to take into account how the other stains in your panel affect the respective minus parameter. Many flow cytometry gates are difficult to define. This is especially true when you’re looking at activation markers within a continuum or accounting for the large data spread that occurs when compensating a 10+ color experiment. The only way to convince reviewers that your gate is in the proper place is by using FMO controls. Here’s why you need to use FMO controls for any multicolor flow cytometry experiment and how to prepare these controls properly.

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