1 – Instrumentation

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…

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.

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…

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…

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.

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 days of microscopy, including the use of the first true microscopes by van Leeuwenhoek and others, scientists have been able to start seeing the finest details of a cell. With the development of the flow cytometer, researchers have been able to explore cellular processes in great deal.  For example, modern…

Here at ExCyte–we are excited for all of the new flow cytometry instruments, reagents, and software you will have access to in 2015. There are so many new and innovative products that we have been testing and looking into for you. These are just a few highlights of the great things coming your way in 2015. I. Instrumentation The field of flow cytometry is seeing various trends in instrumentation–on one end are smaller, more compact and more powerful instruments for the high end needs of researchers, and on the other end are easier to use instruments that make flow cytometry…

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 for the flow cytometer. This will include what excitation sources should be available, how many detectors are needed, including specialized detectors (small particle detectors), as the need for automatic sampling. All these factors will in weigh in on the What are the projected future needs of the users? This is…

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.   One major determination that needs to made is will the cells be purified (via sorting) or is the experiment just analytical. The second step in any flow cytometry experiment is to identify the reagents needed for the experiment.  In immunophenotyping experiments, this involved knowing what subpopulations that need to be…

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) Keep the core stream tight – the tighter the core stream (low differential pressure), the tighter the CV on resulting data, therefore the easier it is to determine best placement of gates for sorting. Tight core streams also reduce coincident events (two cells passing through the intercept at the same…

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 14-18 fluorochromes to over 40+ simultaneous parameters. The advantage of mass cytometer include: ● Increased number of parameters measured ● No equivalent autofluorescence – cells do not contain stable lanthanide ions ● Minimal ‘compensation’, mostly due to oxidation of some metals, which is predictable Some limitations of the mass cytometer…

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 cells survive this journey. But some do not. Here are 10 things smart scientists do to improve their cell recovery: 1. They pre-coat their catch tubes. A smart way to improve your cell recovery is to incubate your plastic tubes with a buffer solution containing protein. This will help reduce/eliminate the…