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I am still convinced that my first cell sorter was possessed. The number of issues that I had with the system remains hard for me to believe, even after all these years.
It had been purchased, in part, from one vendor because the sales rep for a competitor was nowhere to be found. At that time, I admit I wasn’t overly diligent in my research process. Since then, I’ve pinpointed some critical questions that need to be answered before purchasing a new instrument.
At the end of the process, a shiny new instrument will arrive at your facility. Make sure you find time to do a shakedown and validate the system. This is the time to get to know it better, identify quirks and potential issues, and develop training and QC programs. Once your shakedown is complete, you can start adding users and encouraging feedback on the system.

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The flow cytometer is an integral component of any flow cytometry experiment, and special attention should be paid to ensuring that it is working correctly and consistently. As an end-user, the researcher should be able to sit down at a machine and know that it is performing the same way today as it was yesterday and last week. Equally important is that if any changes in instrument performance have occured, the end-user knows how they have been addressed and corrected, rather than letting them fester and potentially affect the results. Quality control measurements can include a variety of targets, such as PMT sensitivity, laser alignment, fluidic stability, background issues, and more.

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Some technological advances are incremental, while others are significant game-changing tools that offer the researcher the ability to significantly improve current assays while allowing for new and novel avenues of research to be performed. With speed, sensitivity, and capacity to spare, the ZE5 fits into the game-changing category. Reduced carryover, increased speed of acquisition, and a large number of parameters all open up new and novel assays while improving the quality and reproducibility of ongoing ones.

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Since the first laser was mounted to create the first flow cytometer, there has been a push for more – more lasers, more detectors, more colors. As a result, today’s researchers require a large number of lasers and detectors to ensure current panels can be run and new, expanded panels can be developed. This can be problematic because, in general, making one decision to improve a cell analyzer can limit the analyzer in other ways. It may seem like an impossible task, but the team of Bio-Rad and Propel Laboratories, collaborated to bring the ZE5™ Cell Analyzer to the market and, with thoughtful design, the Analyzer answers these challenges, resulting in a high-end, easy to use, automated flow cytometer.

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Getting a clear signal with reduced noise is an essential component to good data. Adding a threshold when acquiring flow cytometry data is one way to do that. It reduces the number of events by setting a bar that a signal pulse must clear before it is counted as an event. Depending on the importance of the data, the downstream applications for the data (or sorted cells) will dictate how critical the threshold is. In combination with proper sample preparation, appropriate thresholding will reduce debris and ensure best outcome.

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The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens. This article describes how to set up, monitor, and maintain optimal voltage settings for your flow cytometry experiment.

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Most of the interactions that a user has with a flow cytometer is with the fluidics system, and many of the issues that users will face in troubleshooting problems on the instrument will also be here. Understanding how the fluidics system works on your flow cytometer will help you prevent many common issues, prepare your samples correctly, and protect your data. Here are four important things to consider about the fluidics system in a flow cytometer.

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Flow cytometry is a powerful technique impacting both clinical and research. When looking for a career, flow cytometry can take you many places. An experienced flow cytometrist can find a job in a biotechnology company, academia, a clinical setting, and more. To be successful in the field, it’s important to seek out new educational opportunities and network with your peers. Here are 5 tips that can help you turn flow cytometry into a successful career.

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While cells between 5-10 microns in diameter are typically the simplest cells to sort, quality must still be preserved to prevent sacrificing levels of purity, recovery, and viability. While sorting cells 5-10 microns in diameter does not present a particular challenge compared to other cell types, the standard procedures presented in this article must be followed to guarantee quality sorts, time and time again.

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There are numerous different ways to use keywords in FlowJo and other data analysis programs. The problem is most scientists fail to annotate their data properly and pay the price when they want to repeat their experiments. By taking advantage of the keywords listed in this article and by using keyword formulas, you can save time during your analysis. Most importantly, when you go to reanalyze your data, you can utilize your previous keywords and formulas to save even more time.

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This article is the second in a two-part series outlining some of the major components of the optical systems used in flow cytometry to provide insight and understanding to what happens before a signal is produced from the PMT detectors. Serving as a knowledge toolkit that can help troubleshoot problems you may encounter when performing your next cytometry experiment, this article investigates lenses, mirrors and filters in your flow cytometry equipment.

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Understanding the optical system of a flow cytometer may seem unnecessary for performing a typical experiment, but the more you know about your instrument, the better you will be at understanding your data, as well as troubleshooting potential issues. This article breaks down 4 elements of flow cytometer optics to provide a broad understanding on its impact on fluorescence.

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The last 40 years have seen significant advancements in cell sorting technology. Cell sorting is often the entry point for many experiments. Fluorescent Activated Cell Sorting (FACS) combines the traditional power of flow cytometry and couples it with the ability to isolate the cells of interest. Understanding the inner workings of the instruments and some rules for preparing samples will lead to more successful experiments. Here are 4 essential facts about FACS.

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Implementing a system of quality assurance protocols lends confidence to the data collected, especially for those researchers performing longitudinal studies. Optimal instrument setting, cytometer sensitivity, and monitoring of day-to-day variability in measurement leads to improved assurance for those using this instrument to collect their critical data. QC programs will continue to be prudent measures for cytometrists to take as they align with the current emphasis on quality and reproducibility.

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Fluorochrome emission is the lifeblood of flow cytometry. The use of in silico tools, like spectral viewers, can save a lot of effort and missed opportunity by allowing for the modeling of excitation and emission profiles in the context of what filters a given instrument is equipped with. Using these tools, it is easy to identify where a new fluorochrome will be measured on an instrument, where a fluorochrome may cause issues with other fluorochromes, and what filters are best for detection. These tools can save a lot of troubleshooting at the beginning of an experiment, and also help provide understanding when issues appear.

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Sorting efficiency, in fundamental terms, is a real-time measurement, generated by the instrument, of how successfully its sorting system is able to resolve cells that we want to sort (target events) from cells we do not want to sort (non-target events). In order for the instrument’s sort output be acceptable with respect to the researcher’s needs, it is not sufficient to simply tell the instrument WHAT to sort, but is also critical to tell the instrument HOW to sort the target population. The HOW is determined by the sort modes.

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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.

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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.

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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.

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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.

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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.

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Microvesicles originate from cells and have the same analysis requirements as cells. For these and other reasons, flow cytometry is a popular choice for microvesicle analysis. However, there are pitfalls with small particle flow cytometry that have led to many conflicting publications. The only way to avoid these mistakes is to first identify them and then take measures to prevent them. The following are 4 common mistakes researchers make when preparing microvesicle flow cytometry experiments, as well as how to prevent these mistakes.

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To make certain your instrument is set up correctly for your experiments, manufacturers have developed defined polystyrene beads.

These beads’ consistent nature helps you to assess how your instrument is behaving, helps you set up proper compensation matrices, and helps you generate volumetric counts of your cell populations. Alignment, sensitivity, and fluidic quality control beads will help you to ensure that with the same wattage on the laser and the same voltage applied to the detector returns the same median fluorescence. The right compensation capture beads will bind antibodies of multiple isotypes from multiple species and give you a very bright positive signal from which you can calculate a correct compensation matrix. The use of counting beads allows you to easily calculate your cell concentration in your sample. Together, these beads will make your life easier and help you get your data published. Here are the 3 beads you should use.

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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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With the increased development of fluorescently conjugated monoclonal antibodies came more applications with potential clinical impact.

In bone marrow transplantation, studies using hematopoietic cytokines made it feasible to gather stem cells from peripheral blood. It was also shown that reconstitution of bone marrow was accelerated when using cell from peripheral blood rather than bone marrow. Many more clinical flow cytoemtry applications have been developed. All of which should follow these 6 keys of running clinical flow cytometry experiments.

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Written by Tim Bushnell, PhD What happens if one combines the power and speed of traditional flow cytometers with the resolution of a microscope? Cytometry is the study of biological processes at the whole cell level and includes techniques like light microscopy and electron microscopy. But microscopy by itself is a bit different. From the earliest…

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Written by Tim Bushnell, PhD What is the top flow cytometer? The easy answer is the flow cytometer that matches your needs and fits within your budget. However, before running off to spend cash, consider the following. What are the current needs of the users? Evaluating the user’s needs will help define the parameters needed…

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Written by Tim Bushnell, PhD Flow cytometry is a powerful tool for asking and answering questions at the whole cell level. The first step in any flow cytometry experiment is to define the hypothesis or biological question that is to be answered.  This helps ensure that flow is the correct technique for answering the question.…

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Written by Tim Bushnell, PhD Cell sorting remains the best tool to isolate and purify cellular populations that can be phenotypically defined. This is especially true for rare-event detection and purification. Successful rare event detection and purification requires some attention to ensure the best yield and purity. 1) Watch the instrument to ensure success a)…

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Written by Tim Bushnell, PhD Mass Cytometry, commercialized by the company DVS Sciences, in the instrument called the CyTOF is a newly emerging technology in the field of flow cytometry.  This technology replaces traditional fluorescent-labeled antibodies with highly purified, stable isotopes with very well characterized mass values.  This extends the power of flow cytometry from…

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  Written by Tim Bushnell, PhD Cell sorting can be a scary proposition. A precious sample is introduced into a machine that pressurizes the cells to 70 PSI, moves them past one or more lasers, vibrates the stream at 90 kHz before decelerating the cells to atmospheric pressure before they hit an aqueous surface. Many…

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Written by Tim Bushnell, PhD Cell Sorting is the process of isolating cells after the identification of the cells using the principles of flow cytometry. The upstream components of the cell sorter are common to all flow cytometers. The difference comes in what is done with the cells after they have been interrogated and identified.…

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Written by Tim Bushnell, PhD The question always arises as to what is the top cell sorter on the market. This question is a difficult one to generalize because there are several considerations that need to be made in choosing a cell sorter. What are the sorting needs of the investigators? If all the investigators…

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Written by Tim Bushnell, PhD A laser type in a flow cytometer with a wavelength of about 560nm. The green and yellow laser are more effective at exciting PE and its tandems than the traditional blue laser. The yellow laser is also often used to excite the “fruit” dyes like mCherry. For more information, please…

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Written by Tim Bushnell, PhD The laser type in flow cytometers with a wavelength of around 530nm. Standard “green” lasers are about 532nm, but vary between 530nm and 535nm usually. The green and yellow laser are more effective at exciting PE and its tandems than the traditional blue laser.

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A laser with a wavelength in the UV range. Typically in flow cytometers, the UV laser has a wavelength of 350nm or 355nm. Some have a wavelength of 375nm.

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Another very common laser after the “blue” and “red” laser in flow cytometers. A “violet” laser in flow cytometry typically is referred to as the 405 because most flow cytometers use a violet laser with a wavelength of 405nm. Pacific Blue and Pacific Orange are the most common fluorophores used with this laser, but Brilliant…

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The second most common laser in a flow cytometer after the “blue” laser. The “red” laser typically has a wavelength of 633nm, but new flow cytometers are starting to use a “red” laser with a wavelength of 640nm. The most common fluorophores excited and detected off this laser are APC, Alexa Fluor 660, Alexa Fluor…

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The most common laser type in a flow cytometer. Typically, this laser has a wavelength of 488nm in flow cytometers.  In fact, the term “Blue” laser is often interchanged with “488” laser. Frequently used fluorophores excited and detected by this laser are FITC, Alexa Fluor 488, PE, PerCP, and their tandems.

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A filter that allows light between a set wavelength to pass through and reflects light above and below the set wavelength. For example, a bandpass filter with a wavelength of 550/40nm would allow light between 530nm and 570nm to pass through, but reflect light below 530nm and above 570nm.

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A filter that allows light over a set wavelength to pass through and reflects light above the set wavelength. For example, a shortpass filter with a wavelength of 450nm would allow light with a wavelength less than 450nm to pass through the filter, but reflect light higher than 450nm.

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A filter that allows light over a set wavelength to pass through and reflects light below the set wavelength. For example, a longpass filter with a wavelength of 670nm would allow light with a wavelength greater than 670nm to pass through the filter, but reflect light lower than 670nm.

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Counted by the Photomultiplier Tube (PMT) in the flow cytometer. Photons enter the PMT and the signal is amplified in the PMT when a photon strikes the anode and “knocks” of electrons. These electrons then hit a series of subsequent anodes, amplifying the total number of electrons of signal. The PMT then counts the total…

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A type of flow cytometer manufactured and sold by BD Biosciences. This instrument was one of the first mass produced flow cytometers. The FACSCalibur is still prevalent in many labs around the world. While only a four color, six parameter analog system, this machine is stable and rarely requires service. It has gained a reputation…

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Written by Tim Bushnell, PhD PBS is the acronym for phosphate buffered saline. Phosphate buffer is one of the most common buffers used in biological research.  The phosphate serves as a buffer to keep the pH constant, while the saline is referencing the osmolarity.  Additional ions such as Ca2+ or Mg2+ , energy sources like…

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Written by Tim Bushnell, PhD What is autofluorescence? Autofluorescence is the term given to describe the natural fluorescence that occurs in cells. The common compounds that give rise to this fluorescence signal include cyclic ring compounds like NAD(P)H, Collagen, and Riboflavin, as well as aromatic amino acids including tyrosine, tryptophan, phenylalanine. These compounds absorb in…

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Written by Tim Bushnell, PhD Dynamic range is the total range of fluorescent values obtained from a particular flow cytometry assay. It is defined as the ratio of the largest possible fluorescent signal to the smallest possible fluorescent signal. The dynamic range can vary based on the application. For example, a cell cycle assay may…

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Written by Tim Bushnell, PhD Sheath fluid is the solution that runs in a flow cytometer.  Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure.  The principles of hydrodynamic focusing cause the cells to align, single file in the direction…

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Written by Tim Bushnell, PhD In flow cytometry, cells, in suspension are moved from the tube to the interrogation point and finally into the waste (or to be sorted, but that is a different story).  To do this, the fluidics components of the flow cytometry are required. The fluidics are comprised of a running (or…

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Written by Tim Bushnell, PhD Differential pressure based flow cytometers currently dominate the market. These systems have two pressure regulators. The first is at a constant pressure that sets how fast the fluids runs at. The second is regulated by the investigator (like as shown on this LSR-II control panel). As the sample pressure goes…

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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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