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The most important part of executing a flow cytometry experiment correctly is actually understanding what you are doing. This means you must understand the terms and definitions that are critical to the field of flow cytometry. You must also be able to communicate your methodologies and results intelligently. To this end, we have compiled this list of the top 12 most commonly unknown or commonly misunderstood flow cytometry terms and definitions.

Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation. Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’, is not the correct way to determine proper compensation. The best practices for compensation involve following some very specific rules. Here are 4 steps to correctly compensating 4+ color flow cytometry experiments.

When training new users on data analysis, there are several different best practices and gating strategies you should incorporate into your analysis. There are also several misconceptions you must understand. There are 3 gates that many researchers are not using but should be using when analyzing their flow cytometry data. These gates are critical for good data analysis. They will help remove many confounding events that may be clouding your analysis, especially where rare events are concerned.

Forward scatter detectors collect light at small angles relative to the incident beam and can take advantage of the fact that cells preferentially scatter light in this “forward” direction. Forward scattered light is traditionally and often effectively measured with a photodiode, rather than the more sensitive photomultiplier used to measure fluorescence and side scatter. Scatter gets dim very quickly when particles have diameters below the wavelength of illuminating light, considering that scatter intensity decreases with a dependence on r6 of the particle. Here’s how small particles affect light scatter.

The T-Test compares the differences between the means of two populations to determine if the null hypothesis should be rejected. At a minimum, to perform the T-Test, one needs the means and standard deviations of both populations, and the number of measurements. The researcher also needs to set the threshold value, also termed the α. Then, you will compare this threshold to the P-value. If the P-value is greater than the α, there is no significance in the data. However, if the P-value is less than the α, there is significance in the data. Here’s how to run a T-Test.

From instruments, to reagents, to software there are many flow cytometry innovations to pay attention to this year. This article discusses a few highlights of the great things coming your way in 2016 in the field of flow cytometry.

Methods sections in scientific papers are often unable to capture all the critical data necessary to accurately reproduce the results in another lab. Here, information is provided on two specific ways in which flow cytometry researchers are effectively communicating flow cytometry data and metadata to the greater flow cytometry community to improve reproducibility and consistency. These two ways include first, the use of the MIFlowCyt standard and second, sharing data using the Flow Repository.

Reproducibility is a critical component of the scientific process. One cannot publish data if the experiments cannot be replicated. Unfortunately, as Begley and Ellis pointed out in a commentary in Nature that when Amgen attempted to reproduce 53 “landmark” papers in the area of cancer research, only 6 papers were “confirmed.” What does that mean to you, the flow cytometry researcher? To avoid publishing errors that reviews despise, it’s important to follow and promote the best practices in the field, thus ensuring that your data is reproducible to investigators attempting to validate your research. In particular, you must follow these 5 experimental tips.

T regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens. Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells). Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated. This article will show you how to quantify Tregs and how to ensure you're measuring true suppressor T cells.

The cell sorting process is inherently stressful. Cells are first manipulated in suspension for up to several hours to prepare and stain them. Then, during the cell sorting process, these cells are pushed through narrow tubing under high pressure in the range of approximately 10-70 PSI, rapidly depressurized after passing through a nozzle, and then jetted through the air at velocities of 20 m/s (~44 MPH) or higher. Keeping cells healthy, happy, vital, and viable over the course of a cell sorting experiment is important to keep cells alive during the sort but also that the recovery of cells from the sort is high. Here are three things you can do to help ensure high levels of viability.

The Complete Blood Count is a powerful addition to many flow cytometry workflows. The CBC is an automated hematology test that looks at the levels of all the cells in your blood, providing your physician with valuable information about your health. Using just a small sample of blood, the CBC generates an extensive amount of information WITHOUT the need for centrifugation or multi-color staining experiments. Running a CBC is fast, easy, and inexpensive. In the world of clinical research, a CBC should always be run on the human clinical research samples. As a result, any obvious outliers can be removed from the study, reducing the spread of the data and reducing the risk of confounding your interpretation of the data. Here are the major advantages of obtaining a CBC by flow cytometry.

When setting up a cell sorting experiment, there are many things to consider. You must consider which controls you’re going to use, how you’re going to compensate the experiment, which instrument and which instrument settings are ideal, and how you plan to analyze, gate, and present your data. With so many things to consider, it’s easy to lose site of the small things that can drastically affect the viability of your cells, including the composition of your suspension buffer. The composition of the suspension buffer for preparation, staining, analyzing and sorting is perhaps the most important parameter for maintaining viability during a cell sorting experiment. While the precise components of a buffer can differ depending on the cell type, there are a 5 key points to keep in mind.