Kinetics analyses are performed by selecting a population in the workspace and choosing Kinetics... from the Tools menu. If a gated population is selected, then the kinetics analyses are performed only on the cells that fall within the gated subset.
Kinetics analysis 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, it will use the event number, and the file's begin and ending times to approximate of time. In this case, FlowJo will assume that there was a constant event rate during collection.
FlowJo opens the graph window for kinetics analyses, as shown below. Roll your mouse over the red dots for more information.
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.
This button opens a dialog for creating multiple time slices in one step. Specify a prefix for the names that will be added to the workspace, and either the number of slices to be created, or the duration of each range you wish to create.
Choose a function to be applied to the Y parameter for display. The functions that can be displayed are each calculated on the Y parameter for each time interval across the entire graph. FlowJo selects an appropriate time interval over which to calculate the function values (usually, this value is 1 channel of the time parameter). After the function is computed, any smoothing that has been selected is applied; the result is graphed in the window. The possible functions that can be displayed are:
The remaining functions in the drop-down list are computed based on a threshold value. The threshold is a scale value of the Y axis parameter (i.e., not the raw channel value, but the binned linear fluorescence value). The threshold can either be an absolute value, or a relative value based on time slices that you have defined.
When you create time slices they are displayed here beneath All Events. 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 units for the peak value and the slope are the same as the units shown on the Y axis - linear scaled units for the parameter being displayed.
Click left or right arrow to change graph to previous or next population at this level. Click up arrow to view parent population.
Start time and end time of each time slice.
The time of peak value within each slice.
The peak value itself.
Average value over time slice.
Linear least-squares within the timeslice.
Area under the curve.
The final column contains the elapsed time for each time slice, Delta T.
Click to select and edit a time slice.
Creates three time slices: One before the stimulation break, one as the ratio rises to a peak, and the third after the peak.
Click to draw a new time slice with the mouse. You will then be asked to name the timeslice.
The value that is displayed in the line graph is a function of the Y parameter (the name of 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.
Shown below is the result of analyzing the graph above, starting with Smoothing.
First, the events were smoothed (via Moving Average.) 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 darker horizontal line across the time slice, and press the delete key.
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 apply kinetics nodes to group nodes to perform batch kinetics analysis.
Visit the kinetics question/answer page for an introduction to kinetics analysis techniques.
Download a Kinetics Workspace with Demo Data to try out this platform.
Download the Kinetics Tech Note to print a short (four page) step-by-step guide.
