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…
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 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…
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…
There are several methods for analyzing live, dead, and apoptotic cells by flow cytometry. As cells die, the membrane becomes permeable. This allows for antibodies to penetrate the cells, which can now mimic live cells. For this and other reasons, it’s important to remove dead cells from further analysis during your flow cytometry experiments. For example, let’s say you merely need to generate an accurate cell count. If you fail to remove your dead cells first, you might think you’re seeding 10,000 cells, but in reality only 7,000 of your cells are actually viable. Since the dead cells in your…
8-peak beads, sometimes called “rainbow” beads, are a set of beads in a single vial that contains 8 different populations that differ only in the amount of fluorophore contained within them. One of the peaks, termed Peak 1, is unlabeled, and the additional seven, termed Peaks 2-8, contain increasing amount of fluorophore. 8-peak beads are designed to fluoresce in all channels on most flow cytometers and cell sorters. These beads are used to check fluorescence sensitivity and resolution by measuring the position of the unlabeled peak and the separation between all of the peaks, respectively. They are also used to…
FMO controls are samples that contain all the antibodies you are testing in your experimental samples, minus one of them. When analyzing the minus, or left out parameter in an FMO control, you give yourself a strong negative control to work with. It’s a strong negative control because the left out marker in the FMO control allows you to take into account how the other stains in your panel affect the respective minus parameter. Many flow cytometry gates are difficult to define. This is especially true when you’re looking at activation markers within a continuum or accounting for the large…
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…
Flow Cytometry is a remarkably powerful tool for the study of T cells. It has been successfully used for many decades to accurately visualize and enumerate a variety of T cell subsets. With a large sensitivity range for fluorescent probes, >95% sampling efficiency, and the ability to sort populations of interest for further study, fluorescent-based cytometry remains a tool of choice for T cell analysis. The key is to define your T cell populations of interest with correct gating strategies and to back up your T cell subset findings with functional analysis of these subsets. A cell’s actions should guide…
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…
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.
All the experiments and experience in the world do not count if you are unable to communicate your results to the scientific community. As part of that communication process, your paper will undergo the dreaded ‘Peer-Review’ process. If you wish your paper to survive this process, you must collect, analyze, and present your flow cytometry data properly—before you submit your paper. A review of the following questions, as well as how to answer them, will help ensure your paper is not rejected. Here are 5 specific questions reviewers will ask when reviewing your flow cytometry data.