how the new concatenation works

The goal of this exercise will be to combine 8 tubes of an antibody titration experiment into one file and then display it as Fluorescence vs. tube #. Our Basic Tutorial data set (google "flowjo basic tutorial") will be used.

First, I create some sort of gate delimiting my cells of interest. I don't want to concatenate all the events, just the interesting ones. I called my gate "Lymphocytes" and applied it to the group:

The next thing we will need is a keyword which we will fill with some values in order to identify the tubes. I'll be using "tube number" filled with integer series {1,2,...8}. Other ideas include dilution factors, or dilution concentration of your antibody, etc. - as long as the values are numerical.

Note: if you need a new keyword, click Workspace menu, new keyword. Otherwise use one of your pre-existing ones. You can access those via Workspace/edit columns.

So here are my tubes lined up with tube numbers in the keyword column:

Note: you can quickly make value series. Double-click in the cell which corresponds to the first editable keyword value (could be blank, or filled. ). Then option-click (hold down option) the workspace menu, and choose "create val series". The dialog is self explanatory and helps you create any sort of numerical series you might need for this task.

Next, click any of the "Lymphocytes" gates and click Workspace/Concatenate. Most of the options you'll see in the Concatenate dialog are self-explanatory. The ones to pay attention to:

- Create Additional Parameters - click Choose and pick the keyword which stores values you wish to make into a parameter in your concat output.

- Batch Concatenation - be sure to select the right option and number here! In my example, I picked "concatenate successive files together" and N=8.

Then let it rip.. it'll ask you where to put the output file. Make a new workspace, and drag in your concatenated file. You'll notice the parameter list will now include the tube number parameter, or whatever, so you can graph vs. that.

 

Note: in case of FCS3 data, flowjo will try to recognize if your keyword's values create a patter better seen in log and automatically adopt log scaling. In this example, we're using a series of numbers which are linear.

DD Reader Ben has sent in some of his own usage examples in titration:

Comments

[Catarina Tran]

Hi-

I am having problems with Concatenation. Once I enter my data and export the files to another workplace the FCS file does not show any concatenation. Please help.

[Emilie]

It does not work for me either. The concatenated axis does have any values and my samples are off scale.

[Maciej Simm]

Catarina, Emilie, please remember that the support line is flowjo@treestar.com, this blog is not interactive (it does not notify me when comments are made, for example.)

To answer your question, I will need to see your setup, including your choice of keywords and their values.