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 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. Likewise, a percentile gate of 0 to 5 results in a gate for the dimmest five percent of the events.After completing the perfect staining and cytometry run, the hard work begins – data analysis. For instance, if a selected gate has a lower bound of 50 and upper bound of 100 (for percentile) FlowJo constructs a gate which includes the brightest half of all of the events. When percentile is selected, FlowJo will create a gate that includes events that have greater intensity than the fraction of cells specified by the lower bound but a lower intensity than the fraction of events specified by the upper bound. When this option is selected FlowJo will create gate boundaries that align with the numerical scale on the axes. A percentile is the value of the intensity below which the specified portion of events occur. A control gate will reflect the position of the lowest 5% of the control sample no matter which additional samples it is applied to. Absolute intensity is the scaled intensity value displayed on the axes in FlowJo. For example, if a non-control gate made on a histogram is set with a percentile range of 0 to 5, it will re-size to include the lowest intensity bearing 5% of the cells on each file it is copied to. A control gate will adjust it’s shape on the initial sample based on percentile or intensity, but will remain constant if copied to other samples.
#FLOWJO 10 BACKGATING FULLY GATED MANUAL#
#FLOWJO 10 BACKGATING FULLY GATED PC#
To manually enter a gate from the Graph Window, select the desired parameters using the X and Y axes drop-down selectors, and then choose Manually Enter Gate from the “Graph” menu at the top of your screen (this can also be accessed with command + G on a Mac and control + G on a PC when the graph window is selected). Manual gates are used to set gates by intensity or percentile.