The “Divide by…” choice has been included to accommodate users of cytometers that scale their data down for display in the acquisition software, but record the actual measurements, such as most Beckman-Coulter products. The values recommended by the individual cytometer manufacturers have been implemented here by default, and so in most cases the user will not need to adjust these values. Section 3 allows the user to select whether they would like the scatter parameters to be displayed by default on a linear or logarithmic scale, what the range of all parameters shall be, and whether the data should be scaled. Section 2 identifies the keyword values that FlowJo uses to determine which cytometer has been used. The top choice is “Generic”, and will be used by default whenever FlowJo cannot determine which cytometer was used for acquisition. Clicking on each cytometer shows the display settings for any file identified as having been collected by that cytometer. The red numbers have been superimposed on the window as an index.Ī list of cytometers is on the left of the screen, near the red number 1. Once you click this icon, the window shown in the figure below will appear. To access these settings, click the Preferences button, then from the preference menu click on the Cytometers icon which is outlined in red in the figure below. Through these keywords FlowJo v10 has the ability to discern when acquisition software was used to create each data file, and then apply scaling preferences on a file-by-file basis. They also have the $SYS keyword which lists the software system that was used. All FCS files have the keyword $CYT which lists the cytometer used to collect the data. Keywords that are standard to all FCS files are prefaced by a $. If you cannot get your data to look like it does on the machine – PLEASE contact us right away)Ĭytometers output FCS (Flow Cytometic Standard) files that contain keywords, information about the data organized by three letter codes. (*Please note – Instrumentation companies are constantly releasing updates to their software and do not always notify us of changes they have made to scaling keywords. 2006 69A:541–51.FlowJo version 10 has a new preference setting that allows users to customize the data scaling based on the cytometer used to collect each file. A new “Logicle” display method avoids deceptive effects of logarithmic scaling for low signals and compensated data. A harmonized approach to intracellular cytokine staining gating: Results from an international multiconsortia proficiency panel conducted by the Cancer Immunotherapy Consortium (CIC/CRI). Using the geometric mean fluorescence intensity index method to measure ZAP-70 expression in patients with chronic lymphocytic leukemia. Finally, we will conclude with the fundamental principles of compensation. We will define what CS&T is, why it is necessary, and how to apply all these concepts in basic experimental setup. We will describe the differences between biexponential versus logarithmic scaling. We will also outline how to interpret flow data on commonly used data plots including histograms, dot plots, and density plots. In particular, we will discuss FSC and SSC in detail with emphasis on gating strategies and how dead cell exclusion can be used to clean up your data. In this chapter, we will define basic terminology that will enable end users to understand concepts important for experimental setup and how to interpret flow cytometry data. However, it is not there is just a lot of jargon to learn like in any other field. The language of flow cytometry inadvertently gives the impression that this technique is difficult.
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