When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. What Is A Fluorescence Minus One, Or FMO, Control?įMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them. How are you going to account for the data spread that occurs with compensation? In any multicolor flow cytometry experiment, the answer to your gating troubles is to use Fluorescence Minus One, or FMO, controls. Now, consider gating out subsets in complicated 10+ color experiments. How can you convince reviewers that you didn’t make an error and placed your gate in the proper place? This is especially true when you’re looking at activation markers within a continuum, or dim levels of positivity, like FoxP3 in regulatory T cells. Other gates, however, are more difficult to define. CD19+ B cells are often easy to pick out on a plot showing CD3 for a T cell subset sample. For example, in flow cytometry, some gates are “easy” to set. Valuing your control samples over your experimental samples is a mistake, especially when performing flow cytometry experiments. We’ve all had experience working with someone who wanted to see the “real” samples, not the control samples, first.īut if you’re a smart scientist, you know “real,” or experimental samples are meaningless unless you know, among other things, the background levels you’re working with. Why Fluorescence Minus One, Or FMO Controls, Are Important Your gate placement will be questioned, your samples will be questioned, and your papers and grants will be questioned. Without the proper controls, you would not be able to compensate your flow cytometry experiments correctly or identify your cells of interest correctly.īy using the wrong controls, or leaving out certain controls altogether, you leave yourself wide open for criticism. It’s also important for correctly interpreting your data and drawing correct conclusions. Setting up the right controls is critical to determining how your cells are responding to treatment. However, you must control for these factors. Reagent selection, well-maintained instruments, and other factors are also important. What are the three most important parts of any flow cytometry experiment?
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