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In today’s world, many scientists have access to instruments capable of running experiments with 10 or more colors.

The leap from 2 to 10 colors may seem small, but here are many factors to consider in the design and analysis of experiments that makes full use of instruments that can handle these additional colors. Imagine analyzing a 2-color experiment. With 2 biaxial plots and a single quadrant gate, you have only 4 populations to report. Now add a 3rd color. By doing so, you’ve increased your population count to 8. With 4-colors, you’ve increased your population count to 16. On and on it goes until you get to 10-colors. Now you have 1024 possible combinations! With this kind of complexity, careful experimental planning is not a luxury, it’s a necessity. Here are 7 tips for preparing and analyzing 10-color flow cytometry experiments.

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Flow cytometrists use the Jablonski diagram to aid in understanding and explaining the kinetic events of fluorescence.

Fluorescent compounds start at the ground state until they are excited by interacting with a photon of light. This photon excites the compound, promoting an electon to a higher energy state. Some of this energy is lost by emission of heat and other non-radiative processes, leading to the previous energy state. Finally, an electron falls back to the ground state while releasing a photon of light. This photon has a lower energy (higher wavelength) than the exciting photon of light. Here’s how understanding this process can help you get published.

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