Either forward scatter or side scatter are good choices, as they are both intrinsic measurements of all events passing through the laser intercept. The data are plotted against a time parameter versus a scatter parameter. These plots show the sample running evenly over the time of acquisition. An example of this is shown in the below plots. The principle of this step is to ensure a good and even flow stream during the instrument’s run.Ĭlogging, back-pressure and other instrument-related issues can affect the flow, so eliminating cells that may have been affected by such problems is an important step to cleaning up the data. The details on this hierarchy, including how each fits together sequentially to produce the optimal flow cytometry figure for every experiment, are outlined below… 1. Backgating - to provide visualization of cells in final gate at higher level.This is where Fluorescence Minus One (FMO) controls become critical in defining the populations of interest. Using viability dyes and dump channels further narrow to the cells of interest. Subsetting gating - to rely on expression of markers and what they identify.Forward and side scatter gating - to remove debris and other events of non-interest while preserving cells based on size and or complexity.Pulse geometry gating - to remove doublets from the dataset.Flow stability gating - to capture events once the flow stream has stabilized, eliminating effects of clogging, back-pressure, and other instrument issues.To this end, the following hierarchy was created to help you gate your events correctly… Hopefully, you will objectively choose the right events to display. In other words, you can reuse and refine your gates and plots over and over again without actually losing cells, but you and you alone will determine which events you are displaying. While actual cells will not be lost in trying various gating strategies, data points can be eliminated from your population. 5 Gating Strategies For Publishing Flow Cytometry Data This is also where science becomes an art form. Thankfully, there are many ways to avoid shaping the results, and instead sifting for the real and actual data that is relevant to the flow cytometry experiment at hand.Ĭommunicating the results of a flow cytometry experiment is where the researcher has the power to make new or subtle findings instantly comprehensible to the audience. Science must be objective, or it is simply an exercise in creative sculpting, which does nothing to move science forward. The critical difference between sculptor and scientist is that while the sculptor is guided by a creative vision, the researcher is guided by very particular laws of nature and a specific method of working through a biological hypothesis to avoid shaping the results to his or her whims. There is a story inside the data, and it is the job of the researcher to unravel it. When sitting down to perform a new analysis of flow cytometry data, it is much like Michelangelo staring at a piece of marble. For researchers studying these cell populations, or in cases where light scatter gating does not adequately exclude these cells from the analysis, this background may be an important factor to consider when selecting reagents for panel(s).“Every block of stone has a statue inside it and it is the task of the sculptor to discover it.” - Michelangelo Note: When using high concentrations of antibody, background binding of this dye to erythroid cell subsets (mature erythrocytes and precursors) has been observed.
#Flowjo 10 normalization histogram plus#
563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. This will ensure that BD CompBeads are appropriate for your specific cellular application.įor optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD CompBeads. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. When fluorochrome-conjugated antibodies are bound to BD CompBeads, they have spectral properties very similar to cells. BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).
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