The first step to any successful flow cytometry data analysis is proper gating. For a complete overview of gating terminology, types and strategies, check out our previous Gating Newsletter. Here we outline 6 questions that cytometry researchers should ask themselves to help improve your gating techniques. Questions 1-3 should be asked as the experiment is being designed, while questions 4-6 can be asked during data analysis.

1. What are my populations of interest and how have they been defined in previous studies?

Save yourself some hassle by learning from those who have gone before you! Check the literature for any information on how your populations have been defined in previous experiments. Use this information to help set up your experiment design and to ensure that you are using the proper stains and controls to analyze your data.   “If I have seen further than others, it is by standing upon the shoulders of giants.” – Isaac Newton

2. Which markers will be expressed?

When planning your panel design and gating strategy, it is important to have a strong understanding of which markers will be expressed by your population of interest. Once again, it may be helpful to review past publications for useful information on your cell types. Be sure to consider disease states and environmental conditions that may affect marker expression levels. “Express Yourself!” – Charles Wright

3. Am I using the proper controls?

Including all the necessary controls for your experiment type is critical to successful gating and data analysis! These typically include viability dyes as well as FMO, internal negative, unstimulated, and isotype controls. Learn More about the different types of controls to consider for your next experiment. “Only you can control your future.” – Dr. Seuss

4.Was my flow rate stable?

Poor flow rate during your experiment can cause artifacts in the data. Eliminate these by plotting time vs scatter to determine how stable your flow was during the cytometry run. A good flow rate will plot as a single flat line, whereas poor flow can be identified as uneven spikes and valleys. “Be still like a mountain and flow like a great river.” – Lao Tzu

5. What size are my cells?

Using forward and side scatter gates to exclude cells based on size can be useful, but also dangerous. Be sure to consider all cell types in your sample, including any cells known to change size under varying conditions. Also, ensure the samples you are analyzing are individual, living cells by excluding doublets/clumped cells via pulse gating and dead cells/debris via viability dye and FCC gating. “It’s not the size of the dog in the fight, it’s the size of the fight in the dog.” – Mark Twain

6. Do I have appropriate Analysis Software?

There are many commercially available analysis software options optimized for different hardware or specific types of analysis. Find a complete list on our analysis software page. “Software is a great combination between artistry and engineering.” – Bill Gates. Asking yourself these six simple questions can save you a lot of trouble with your data analysis.