Tips for how to perform certain kinetics analyses.
This list is not comprehensive; you should read the main Kinetics help page 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 Statistic 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 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 time slice (thus, the background time has 5% responding cells.) You might also choose the 98th percentile (thus limiting responding cells during the background to 2%).
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 samplerather, FlowJo calculates it with respect to the particular time-period you define as background.
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?
You can copy from the Kinetics window and paste data into a spreadsheet or similar program. In the Edit menu select ‘Copy Time Series’, then paste into the desired program.
Note that if you have selected smoothing in the main Kinetics window, then smoothed values are copied. In other words, whatever values were used to plot your graph will be pasted to the spreadsheet.
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