Light is used to illuminate the cells in the channel.Sample cells are passed through a narrow channel one at a time.clusters of differentiation or CD markers) can be used to better identify and segregate specific sub-populations within a larger group. When additional information is required, antibodies tagged with fluorescent dyes, and raised against highly specific cell surface antigens (e.g. For example, in immunology flow cytometry is used to identify, separate, and characterize various immune cell subtypes by virtue of their size and morphology. Flow cytometry is a particularly powerful method because it allows a researcher to rapidly, accurately, and simply collect data related to many parameters from a heterogeneous fluid mixture containing live cells.įlow cytometry is used extensively throughout the life and biomedical sciences, and can be applied in any scenario where a researcher needs to rapidly profile a large population of loose cells in a liquid media. Originally developed in the late 1960s, flow cytometry is a popular analytical cell-biology technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Custom Recombinant Antibody (rAbs) Services.Annexin V-FITC Apoptosis Detection Kits.Also, for some samples such as blood or bone marrow, gating on SSC vs CD45 or other markers might help identify the cells you need.Įssentially, it is important to play around with the data and check everything, as your cells may not be where you think they are. In this case using a viability dye to remove debris and dead cells may be useful, as would backgating (discussed below), to identify cells that fall outside of the gates. When gating, bear in mind that blasting cells may be larger than resting cells therefore, tight gates may mean you are missing a population that is important to your experiment. They are often found at the bottom left corner of the FSC vs SSC density plot (Figure 2). This gating strategy can also be used to exclude debris as they tend to have lower forward scatter levels. Figure 1 shows how FSC vs SSC gating can be used to identify the distinct cell types in red cell lysed whole blood. Furthermore, for small samples, the level of light scatter does not always correlate with size.ĭespite these caveats, in samples with multiple cell types, this first level gating method is useful for identifying the cells of interest. For example, FSC vs SSC gating is most useful for blood samples but even then, granulocytes (12-17 μm) can sometimes appear larger than monocytes (20-25 μm). While these are an indication based on light refraction, it depends on the sample, the sheath fluid and the laser wavelength. However, it should be noted that forward scatter does not necessarily relate to size and side scatter is not really granularity. It is often suggested that forward scatter indicates cell size whereas side scatter relates to the complexity or granularity of the cell. Our recent webinar on flow cytometry controls provides information on what controls to include in your experiment.įorward and side scatter density plots for identifying your cell population of interest and excluding debrisįorward versus side scatter (FSC vs SSC) gating is commonly used to identify cells of interest based on size and granularity (complexity). Before beginning, you should also ensure that you include proper controls for accurate data analysis. If you have no prior experience with your cells of interest, it is important to check the literature as a guide. For example, the fixation and permeabilization processes during intracellular staining can alter cell size and granularity, resulting in modified forward and side scatter profiles (discussed below). Things to determine are the relative expression levels of cell specific markers, the approximate size of the cells, and whether their size can be affected by experimental conditions. Therefore, before you begin your analysis, it is important to first find out as much as possible about the cells you are analyzing. Your gating strategy is informed by what you know about your cells of interest. Learn as much as possible about your cells of interest So what gating methods do you need to know to confidently analyze your stained samples? This blog post will take you through the various gating strategies for effective flow cytometry analysis. This process of gating can appear quite random to a flow cytometry novice but it is in fact the most important part of flow cytometry analysis. Flow cytometry analysis typically begins with creating gates to distinguish cells of interest.
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