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There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

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There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This blog will focus on recommendations for electrostatic sorters.

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What is the single-most important feature of a flow cytometry experiment? Arguably, it’s the stained cells that gather data about biological processes of interest. However, a flow cytometer can measure cell-like particles as well as cells, which opens the realm of cytometry to the use of microspheres. Most researchers are familiar with the 4-Cs that beads can be used for: Control, Calibration, Compensation, and Counting. Beyond the 4-Cs, many are familiar with the multiplex bead assays for measuring analytes. Today, we will take a look beyond these well-known uses and discover the myriad applications of the “Mighty Microspheres.”

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Because mass cytometry allows users to characterize masses so effectively, data can be normalized much more efficiently than what traditional fluorescent flow will permit. If there is no working CyTof at your institution, you can still partner with CyTof-friendly research institutions that have the technology on hand. And because the samples are fixed, you can ship them overnight. This way, they will be analyzed for you. Today’s article will summarize the functionality of mass cytometry technology. This tech has been commercialized largely by Fluidigm in the CyTof systems. There are 5 key points to cover, or takeaways, that cytometrists should keep in mind as they perform their research. The 5 points include how mass cytometry works, panel design, proper sample preparation, data analysis, and imaging mass cytometry.

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A lot of the troubleshooting is focused on fluidics issues. If you sit down and think about your workflow, and how you might want to add a couple of little tweaks here and there which will ultimately help you improve the quality of your data as well as aid you in identifying issues before they become problems your troubleshooting will be much smoother. Consider these three things, what do you before you start collecting data, ensure you have appropriate plots of time vs fluorescence for each of the lasers your using and apply appropriate gating procedures.

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For those new to flow cytometry, compensation is confusing at best and terrifying at worst. Likewise, those who have been doing flow cytometry since the analog ages may be holding on to practices that, while suited to the analog instruments, should be left to the annals of history. As such, a lot of time is spent discussing compensation and the best practices for this critical process. There are 3 rules that guide proper compensation, and they’ve been written about extensively since they first appeared in the “Daily Dongle” in 2011. Here, we will review the classic rules and expand upon the tacit assumptions required to fulfill them.

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Congratulations, your grant has been funded! Next comes generating data and publishing papers. What was that hypothesis again? It must be in the grant somewhere, right? To avoid even the appearance of HARKing — Hypothesizing After The Results Are Known — it is important to start the statistical analysis process even before the first experiments are performed. This process consists of 5 steps: setting the null hypothesis, establishing a threshold, performing the experiments, performing the statistical tests, and communicating the results. Walk with us as we discuss these steps in an example workflow.

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Dyes exist for the detection of everything from large nucleic acids to reactive oxygen species, and from lipid aggregates to small ions. Concentrations of physiologically important ions such as sodium, potassium, and calcium can be important indicators of health and disease. Calcium ions play an especially critical role in cellular signaling. As a signaling messenger, calcium is involved in everything from muscle contractions, to cell motility, to enzyme activity. Calcium experiments can be very informative, and with the advent of cheaper UV lasers, more and more researchers can use ratiometric measurements to evaluate the signaling processes in phenotypically defined populations.

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There is a lot of preparatory work that must be done before the first flow cytometry experiment can be attempted. Each step builds upon the previous one and extends where the assay is going. Be prepared for some trial and error in this process, and don’t expect perfect results the first time around. An educated user is a good user, and makes the SRL staff’s job that much easier. The partnership between investigator and SRL staff is a rewarding one, when both parties work together to achieve the ultimate goal of generating excellent data and sort results that help answer the biological question being tested.

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Here, we cover 5 lessons from the trenches of flow cytometry looking at important aspects of how best practices have changed over time, which practices need to be adopted, and which are outdated. Put those old, coffee-stained protocols away and take advantage of the best practices for digital instruments to write new and improved ones (coffee stains optional). Your data will thank you.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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 downstream experiments. Here’s how cell sorting droplets are charged and how cell sorting drop delays are determined.

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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 the ICCE will be when flow cyotmetry job postings in basic research start asking for the certification. This day may not be too far away.

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Written by Tim Bushnell, PhD 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…

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Written by Tim Bushnell, PhD I’ve been in the world of flow cytometry and cell sorting for a very long time. Now, don’t worry, this isn’t going to be some lament about the “good old days.” Well, maybe just a little. But there will be helpful takeaways, I promise. I was trained by the incredible…

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Written by Tim Bushnell, PhD As with all complex technology there are many different levels of education that users should avail themselves of. Basic instrument operation This level of education is akin to learning how to drive a car.  At this level, the focus will be on how to put the sample on the instrument,…

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Written by Tim Bushnell, PhD Flow cytometry can be an intimidating tool for the new cytometrist.  There are many sources that one can turn to for training and education. Local Shared Resource Lab Manager If you institute has a shared resource lab (a ‘core facility’), look in what training they provide.  This is your first…

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An implementation of biexponential scaling published by the Herzenberg lab at Stanford. The biexonential scale is a combination of linear and log scaling on a single axis using an arcsine function as its backbone. The “logicle” implementation of biexponential was implemented in many popular software packages like FACSDiva and FlowJo. Other types of biexponential scaling…

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Written by Tim Bushnell, PhD Flow cytometry is a complex technology that requires understanding of sample processing, data acquisition and data analysis.  An individual experiment can take a dozen hours to prepare, hours to collect and days to analysis.  This is why flow cytometry training is critical in understanding and optimizing the use of this…

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Written by Tim Bushnell, PhD If you’d like a job in a flow cytometry core or lab, ExCyte can give you all of the training you need. Combined, our instructors have over 100 years of flow cytometry experience. If you are interested in researching available flow cytometry jobs, there are number of online resources that…

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Written by Tim Bushnell, PhD Individuals can now be certified in flow cytometry by taking the International Cytometry Certification Exam, which is jointly managed by the International Society for the Advancement of Cytometry (ISAC) and the International Clinical Cytometry Society (ICCS).  This exam covers the general principles of cytometry in a multiple-choice format.  Individuals who…

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Written by Tim Bushnell, PhD Every fluorophore has a unique excitation and emission profile which is usually displayed on a spectral viewer, or spectral graph. The combination of the excitation and emission profiles is the fluorophore’s spectral profile. Every fluorophore has a peak excitation wavelength (the wavelength at optimal excitation) and a peak emission wavelength…

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Written by Tim Bushnell, PhD The Fluorescence Minus One Control, or FMO control is a type of control used to properly interpret flow cytometry data.  It is used to identify and gate cells in the context of data spread due to the multiple fluorochromes in a given panel. An FMO control contains all the flurochromes…

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Written by Tim Bushnell, PhD Do you know what an isotype control is? Isotype refers to the genetic variation in the heavy and light chains that make up the whole antibody moiety. In mammals, there are 9 possible heavy chain isotypes and two light chain isotypes. Every antibody will have a specific isotype, and this…

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Written by Tim Bushnell, PhD Titration is the process of identifying the best concentration to use an antibody for a given assay. While the vendor will provide a specific concentration to use, this may not be appropriate for your assay. Performing titration is a simple process: fix the cell concentration, the time of incubation, the…

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Written by Tim Bushnell, PhD ExCyte chooses to train people in flow cytometry because we know what it’s like to feel the pain of ruined experiments. No one enjoys wasting thousands of dollars on reagents and priceless amounts of instrument and personnel time. This is especially true when grant funding comes into play. No one…

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Written by Tim Bushnell, PhD Flow cytometry education has grown phenomenally in response to more sophisticated instrumentation, growing demands for more sensitive, high-speed and multi-parameter flow. Specialized training is critical to any flow lab competing in today’s global marketplace. The key is to find the right trainer. As a core director or lab manager, how…

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Written by Tim Bushnell, PhD A Jablonski diagram illustrates the electronic states of a molecule as well as the transitions between them. These states are arranged vertically by energy and grouped horizontally by spin multiplicity. Nonradiative transitions are indicated by straight arrows and radiative transitions by squiggly arrows. The vibrational states of each electronic state…

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