5 – Flow Cytometry

From photon, through conversion from light, into an electron, into an electronic signal pulse. The journey of a photon, through to its data representation via flow cytometry, involves the detection system and the electronics, and basically following a bouncing photon to its ultimate digitization. This article provides 3 key takeaways in the process from photons through to end data for analysis.

Flow cytometry is a very powerful tool and can answer many questions if the experiments are properly designed. There is a learning curve that takes a bit of time, patience, and practice, but soon you may be finding excuses to perform flow cytometry experiments and we will be here to help you with best practices. Using this checklist will help you to design and perform consistent experiments every time.

T cell differentiation and effector function regulation is an area that needs to be better understood. Until then, speculation combined with best practices help you determine your T cell population’s status. Functional profiling is the primary determinant with surface markers as supporting evidence to assess whether your T cell is activated, resting, or naïve. Here are ways to help differentiate your T cells.

Poorly designed panels, failure to plan for the end-results, and not taking into account instrument characteristics, can all result in a failed sort, lost opportunity, and delay in the necessary data. Take the time at the beginning, before starting a rare event sorting, to understand the different issues that will potentially impact your outcome, and develop a plan to address each of them. Here are 4 considerations to keep in mind.

Fluorescence compensation is one of the more difficult, understandably confusing, and misunderstood aspects of flow cytometry. Understanding what compensation is, why it is necessary, and what to expect when using it, are critical for generating useful and high-quality data from flow cytometry experiments. The definition and mechanics of flow cytometry compensation and the critical concept that compensation’s most basic principle relies on are discussed here.

Every experiment has the goal of ensuring consistent and reproducible data. This makes the proper use of controls to establish the boundaries of gates critical. With the exception of one controversial control discussed in this article, each one of these gating controls plays an important and specific role toward the goal of reproducibility. Using these gating controls in every experiment will reduce data variability within the experiment, as well as between labs and institutions.

Multicolor sorting experiments can be complicated and if not setup properly, result in wasted time and suboptimal results. When setting up a multicolor experiment, the most saliently critical step is to set PMT voltages properly. In addition, using a viability dye and addressing doublet discrimination and setting the right sort regions and gates is important for any kind of flow cytometry experiment, but particularly for cell sorting. These tips help to ensure your setup is perfect to achieve results of the highest caliber.

Cell sorters have become more sophisticated to rival the multicolor capabilities of analytical cytometers with cell sorting experiments becoming more complicated to match. While multicolor sorts are very feasible and can yield excellent results, success is always a product of very careful planning and optimization. From choosing optimal fluorophores to strategies to reducing spillover spreading, this comprehensive article is broken into two parts to give you all you need to play your experiments for advanced accuracy.

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.

Electrostatic cell sorting is a complicated process that continues to be improved. It can be a struggle to understand exactly how all of the sorting components coalesce to accomplish the cell sorter's tasks. For many scientists, the most difficult parts of the sorting equation are how droplets are charged and how drop delays are calculated. By understanding these two things, you will be in a better position to set up a successful fcell sorting experiment, which will help you achieve high sort recovery values, allowing for the accurate analysis of your cells and more cells to work with for your…

The International Cytometry Certification Exam was developed over a period of several years. The goal was to ensure a base level of flow cytometry knowledge in certificate holders. Some cytometrists have deemed the ICCE as unnecessary. Others have voiced concerns about the specialization of certain exam subsections. However, despite these concerns, the ICCE is here to stay. The exam and certification process as a whole has the support of multiple companies that are providing training, as well as the support of ISAC, ICCS, and the Wallace H. Coulter Foundation. In the end, the most telling test of the value of…

We all know that flow cytometry makes individual measurements on large populations of cells, it allows for statistical analysis of the data, lending strength to a researcher’s conclusions. Likewise, the isolation of very complex populations by flow cytometry cell sorting can help lead to a richer understanding of the intricate biology at the genomic, proteomic and functional level. As a reviewer of papers and grants, I am always especially interested in the details of HOW the experiments were performed because that is the critical foundation for what the data is able to tell us–and what it can NOT tell us.…