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After reading this, you might want to look at the kinetics question/answer page for a quick introduction on kinetics analyses techniques. Kinetics analyses are performed by launching the kinetics platform on any population in the workspace-simply select the "Kinetics platform" menu item from the workspace menu. If a gated population is selected, then the kinetics analyses are performed only on the cells that fall within the gated subset. Kinetics requires that there be a parameter collected with time information; and that this parameter is named "Time". If FlowJo doesn't find a time parameter, then it asks if you would like to create a derived parameter that corresponds to time. In this case, FlowJo will assume that there was a constant event rate during collection. The time parameter is created via the "Derive Parameters" dialog, which you can also select directly from the workspace menu. Once you have defined the Time parameter, you can continue. FlowJo begins kinetics analysis by showing the graph window for kinetics analyses, as shown below. This window shows the information and controls associated with kinetics analyses.
Since Time is always on the X axis, you cannot change the x axis. However, you may select any parameter to be displayed on the Y axis. FlowJo, by default, will display a ratio parameter (if one exists); otherwise, it will choose one of the collected parameters. The value that is displayed in the line graph is a function of the Y parameter (this function is shown directly above the graph). In this case, the line graph is the 75th percentile of fluorescence for the "Ca" parameter as a function of time; the line is smoothed using a Gaussian smoothing algorithm with a width of 3 seconds, and auto-scaling is on. To change the function of the Y parameter that is displayed, use the kinetics tools floating window. Activate this window by clicking on the "i" button, or selecting "Graph info..." from the Graph menu. Refer to the documentation about the floating window to see what other kinds of functions you can display. Below the graph is a table of statistics regarding the computed parameter. Each line in the table is a "timeslice"; by default, the first timeslice is "All Events", with a begin and end time that covers the entire collection. The statistics computed for events within a time slice include the peak time and value and the slope of the line within a timeslice. The units for the peak value and the slope are the same as the units shown on the Y axis­linear scaled units for the parameter being displayed. You can copy this information directly to a spreadsheet or word processor: click on the Clipboard button just above the left edge of the table, and a copy of the table is placed on the Macintsoh clipboard. If you switch to a spreadsheet application, select "paste" and the table will be copied in to your spreadsheet. To create a new timeslice, simply click and drag within the graph; much like creating a histogram gate. You will then be asked to name the timeslice; you may also fine-tune the start and end times in the naming dialog window. Shown below is the result of analyzing the graph above: first, the events were smoothed (via the kinetics floating tool window); then, three timeslices were defined to cover the background, response, and resolution time periods. Note that the statistics below the graph reflect these new timeslices, and pertain only to the events within the timeslices. If you wish to delete a timeslice, select it by clicking on the horizontal line embracing the time slice, and press the delete key. (Use option-delete to avoid the confirmation query).
Note that the statistics always reflect the smoothed data: thus, by changing the smoothing parameters, you will affect peak time and value. (The slope is generally insensitive to smoothing). When you select "Copy" from the kinetics window, two different items are placed on the clipboard. One is the graph itself (hold down the option key to include the timeslices as shown in the window). This is what you will see when you subsequently paste into a drawing program. In addition, FlowJo puts the actual kinetics data itself on the clipboard. Should you choose to paste into a spreadsheet, you will get two columns of numbers: the first are the time values, and the second are the computed kinetics values as shown in the graph. This way, you can perform more complex time-series analyses in other statistical packages. Note that these values are the smoothed values, if smoothing is selected from the kinetics tools floating window. Kinetics nodes (shown in the workspace with a "t" icon) behave like other nodes: you can drag & drop them to copy and apply them to other populations. When you copy kinetics analyses to other nodes (in the workspace), all of the timeslices are copied as well as the specific information regarding smoothing, parameter selection, etc. You can update existing kinetics analyses for other nodes to be similar to one you have just modified by dragging the newly modified kinetics node onto the original population: FlowJo will ask you whether you want to replace the contents of the existing nodes; select "Yes". You can attach kinetics nodes to group nodes to perform batch kinetics analyses. See the "Tips" page for kinetics analyses. |
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