FlowJo v9 Manual


Kinetics Tips

Here are some quick answers for how to perform certain kinetics analyses. This list is not comprehensive; you should read the main Kinetics help pages to understand how the platform works before you examine these tips.

How can I determine the fraction of responding cells?

The fraction of responding cells is defined as the percentage of events with a fluorescence (or ratio) above a certain threshold value. You can set this value manually; choose to display % of events over threshold under Function in the Kinetics Options box, then click on Set New Threshold. Enter the exact value you want to use as a threshold.

Another strategy might be to define a certain time period of the analysis as the "background" time. This might be the first 30 seconds of collection; perhaps it is the end of the collection period. Create a timeslice corresponding to the background period. Then, after clicking on Set threshold in the Kinetics Options box, select that time slice in the popup menu. Now choose which percentile of the events in that time slice to set as the background. For example, if you choose the 95th percentile, you ask FlowJo to define the threshold as the 95th percentile of events in your timeslice (thus, the background time has 5% responding cells.) You might also choose the 98th percentile (to limit to 2% responding cells during the background).

The advantage of the second strategy is that when you copy the kinetics analysis to other samples, you don't have to adjust the threshold manually for each sample�rather, FlowJo calculates it with respect to the particular time-period you define as backgroud.

I have determined that only 40% of my cells respond; how do I display the fluorescence of only the responding cells?

Here you have two options. First, realize that the median fluorescence of the top 40% of a population is the 80th percentile of the entire population. Thus, you could display the 80th percentile as a function of time, and you will get a plot that gives you, essentially, the median fluorescence of the responding population as a function of time.

Alternatively, you could define a response threshold (see above), and then choose to display the mean or median of events above this threshold.

I wish to perform more complex statistical analyses on the kinetics data using other programs. How can I get the kinetics data?

Simply copy from the graph window and paste into your spreadsheet. Note that if you have selected smoothing, then smoothed values are copied. In other words, whatever values were used to plot your graph will be copied to the spreadsheet. (Note that there is a Preference that controls whether or not the data values are copied to the clipboard in addition to the graphic; the default setting is to copy both.)

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