![]() ![]() Use the gating tools (top) when you want to create a new gate of a particular kind. Inside the box are any statistics already applied to the population. Above the white box is the number of events gated, the total population, and the Frequency of total.Click this box to choose a color for the transparent overlay.Check here to add a transparent overlay to the gated events.Check here to make the gate adjust automatically to the density of events.The gate can be inclusionary (checked) or exclusionary (unchecked.).Select the type of graph you wish to display.This window, the bottom three sections are hidden click on the black triangles to reveal them. From this window, you can modify the attributes of the current graph (or currently-selected gate). The left plot shows good, even flow, while the right plot shows poor flow.When you open the Options disclosure triangle at the bottom of the graph window, the options below are shown. Using a plot like this will help eliminate artifacts caused by poor flow. Plot a time vs a scatter plot to see how even the flow was during the run. This will clearly eliminate both dead cells and debris from your analysis. It is best to use the scatter gate to remove the debris on the left size of the plot, as well as the small, pyknotic cells that are often FSC small and SSC complex . Blasting lymphocytes are larger than resting cells, and can be missed if there is a tight forward vs side scatter gate. The reliance on forward and side scatter gates as a way to identify lymphocytes from other cells can be rife with peril. Check this first to ensure the proper stains are being used, and the proper controls are in place to analyze the data. How these cells are identified in the literature, or by past experience should guide the experiment. While it may sound flip, knowing what cells are the target of the experiment are critical. Before beginning, know as much as you can about the populations of interest. Cells inside the gate move to the next checkpoint, while cells outside the gate – even by a pixel, are excluded. Gating is an all-or-nothing data reduction process. To properly identify the cells of interest, it is critical to pull together knowledge of the biology with the controls run in the experiment to properly place the regions of interest that will be dictate the final results. After completing the perfect staining and cytometry run, the hard work begins – data analysis. ![]()
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