This question comes up increasingly often in tech support:

- I want to calculate the total fluorescence (integral) of my gate. How do I do it in FlowJo?

as with everything, there is the right way and the quick way.

the **right way** is to take each event's fluorescence and add it up:

- open your population in a graph window as a histogram of its fluorescence

- copy it as text from Edit menu and paste it in excel

- this will create 3 columns: bin number, fluorescence level of that bin, and number of events in that bin.

- make a 4th column multiplying columns B and C across the whole spreadsheet. Then, sum that column.

The sum value is your integral.

Rinse and repeat for other samples.

The **quick way** is to multiply the **Mean** of your gate's fluorescence by **Event Count** of your gate. This is slightly different from the above method, but in my tests, the calculated difference is less than 1%. It is also manyfold faster.

With this gating structure, if we wanted to calculate total CD3 signal in the CD3+ gate, we'd multiply 38 by 4540.

We can automate this calculation for all samples by using the table editor:

Drag the **count** and **mean** nodes to a table, and give them custom names in the "custom" field (over-writing any existing labels). Then add a formula (**fx** icon) and multiply the two stats as shown above. Click the "insert reference to column..." pulldown to find your custom-labels , and separate them with the multiply "*" character.

Then, your integral will be it's own column in the table:

If you use the **PC version 7**, the copy/paste of a histogram into excel only yields 2 columns - bin number and number of cells. You will have to extrapolate the "fluorescence" value of a bin. First, figure out if the bottom value of the bin number corresponds to the right-most value of your histogram's X axis. If it does, you can just do the multiplication like above.

If it does not (ie. histogram goes to 262144, but in excel, it's only 4096) then you have to create another column in your spreadsheet and multiply each bin number by the scaling factor. In this example, the scaling factor is 262144/4096=**64**

If you are not sure what the max value of your histogram is, right-click on the file you're analyzing in the workspace, and select Sample Properties. In the new window that comes up, look at the table "parameters and stains". Third column is labeled range ($PNR) - this will have your original max channel value as reported by the cytometer.

Do the colors in a scatter plot represent the number of fluorochromes used?

Posted by: Steven Wanyee | June 04, 2007 at 05:55 AM

Steven,

the colors in the pseudocolor plot (assuming that is what you meant) represent the density of the events. Blue are the last 5% of density, red are the first. Roughly.

Posted by: maciej simm | June 13, 2007 at 11:11 AM