If you want accurate results, positive and negative carriers must have the same background. The lines connect the positive bead median with the medians of the negative populations. Figure 1 shows the impact of mismatched controls.įigure 1: Unstained cells (green) and unstained beads (purple) are matched with positively stained beads. What you don’t want is to use cells as a negative control with beads as your positive control. However, if you have a mixture of beads and cells, then you must avoid a universal negative. As an example, if you were to use beads as a positive control in your experiment, the correct comparison would be unstained beads. If you only have beads, or you only have cells, then a universal negative can be used without issue. The “universal negative” refers to a process in which a single tube, consisting of unstained cells, sets the negative population for establishing the compensation matrix. Incorrectly employing a universal negative Avoid the following practices at all costs. Now that we’ve covered some proper methodology, let’s get to the 3 faulty methods that will bring confusion and inaccuracy to any flow cytometry experiment. 3 Mistakes That Negatively Impact Flow Cytometry Experiments Otherwise, you’re dealing with different spectra, and you won’t be able to substitute one for the other. You’ll want a matched fluorochrome, matched sensitivity, and matched treatment. Finally, compensation color has to exactly match the experimental color. When a signal violates either of these conditions, accurate compensation becomes impossible.Īdditionally, to properly calculate slope, the positive and negative backgrounds must be the same – otherwise, you’re lacking a control setting for accurate comparison. Remember also that signals need to be on-scale and within the linear region of the PMT detector. Make sure the controls are bright and treated the same way. Thus, the control has to match the sample’s brightness. If the particles are too dim, the resulting slope value will fall well below where it ought to be. Compensation color must precisely match experimental colorĬompensation ultimately is the calculation of the slope of the line between a single stained positive and an unstained negative.The backgrounds of the positive and negative carriers must be matched.Your control must be (at least) as bright as your sample.As you perform your own experiments, remember these founding principles: To start, let’s quickly cover a few ground rules for good compensation. Today’s blog will review 3 incorrect concepts that continue to circulate around this important process and why you want to avoid them. Science is ever a work in progress, and traditional methods are not always the right methods. In fact, it is the longtime cytometry expert who must check themselves for any sort of faith in faulty old practices. And in a topic as dense as flow cytometry, it’s important that we refresh ourselves on some of the fundamentals once in a while. Compensating poorly leads to false conclusions, and you certainly don’t want that.Įven among us flow cytometry veterans, a strong foundation is occasionally in need of a tune-up. Good compensation requires that you tightly adhere to certain rules, understand the function of your instrument, and keep in mind how fluorescence occurs. This is one of the most important parts of the experiment to get correct, and yet there remain rumors and myths that circulate among users that will prevent you from getting correct compensation. Yet even the most talented rookie researchers may find themselves at a loss when it comes to this topic. Putting it basically, compensation is the mathematical process of correcting spectral spillover from a fluorochrome into a secondary detector.
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