So to access your layout editor, you want to click this L button up in the static toolbar. command L is the hot key. It'll open up a new window. It has its own ribbon layout in the header with all of the tools in the layout editor. You can make new layouts by clicking the plus button, delete them with the minus button, or duplicate them in-between. There's also zoom features over here, but what I'm gonna do is drag populations and statistics into this layout editor and then take a look at the gating tree associated with those samples. What I'm gonna start with is my LD1 no stem sample, and I'm just gonna bring it in. Here is LD1 no stem, here's the gating tree on it in my workspace, and I'm gonna grab the CD8 positive T cell gated population o and drag it into the layout. When I drop it there it creates a graph plot in that layout editor displaying that information where I last left that gated population. The last time I was looking at it I was looking at CD8 versus Perforin in that profile.
There are many options to format this graph plot. Some of them are available with a right click. If I right click on the graph plot in the layout editor I get a dropdown menu of things that I can do including showing the ancestry of this gated population. If I click on the ancestry option it will show me the thumbnail images next to that graph plot and all of the gates used to define the population that I'm looking at here. Here's my singlets, lymphocytes, CD3 be positive, quadrate one gates to get to my CD8 positive subset.
You can also do things like show adjunct histograms. That's the one dimensional histograms on the edges of the two d plot. I'm going to make this gating horizontal. Move it over here. There's some information in a text box there below that just describes the sample that we're looking at. What I'm going to do here is actually display a couple different plots and then we'll batch through the samples here. There are page breaks, you can move them out of the way or go to the edit tab and toggle page breaks off. I usually like to duplicate instead of dragging a new one in. Just right click and select the duplicate option that will give me a copy right there and I can delete all the extra information that I don't need.
The easiest way to change the display parameters is to right click on the name, the label of the x and y axes and you'll get that dropdown parameter list where you can then change what distribution you are viewing. I'm going to change this one to pERK and then I'll duplicate this again, move this on over and change the Perforin to Interferon Gamma in the middle so now I've got three plots all looking at CD8 versus something and the gating tree that was used to get to this CD8 positive subset.
Now, one of the coolest features of FlowJo, especially if you're doing lots of samples is the ability to batch report through sample files to display a complex set of plots. Here I've made this layout that shows all my gates and all the responses in my assay. Now I want to take a look at each sample file and batch through here and maybe send a report to my manager or my boss to look at. The first thing we'll do is go this iteration options in the middle of this first layout editor tab and you can see that we have a group that is specified and a iterate by option. The iterate by option is set to off. That simply means that I can bring in any data from any gating population and display it here in a graph plot.
If I want to scroll through all of the samples in a group then I can change this iterate by option from off and set it to sample and that will give me a dropdown list of all of the samples contained within the group I have selected here. My master gates group has all my FMOs, has all of my LD samples and I can start scrolling through them to the no stem but if I do the 20 minute stem there's pERK and you do the two hour stem then there's pERK plus Interferon Gamma. I can scroll through and see the responses of all the markers now step by step in response to the different stimulation conditions.
Then I can generate a report that is external to FlowJo by selecting a type of report here from the next batching menu and then clicking the cog button. Actually, if I want I can just make it in a new layout in FlowJo but what I want to show you is maybe making a webpage or a PDF report if you select the type of report from the dropdown in the batching options. Select the destination location. I'll put it on my desktop and then click this cog button and what FlowJo's going to do is go through the master gates group and make a report showing these plots for every sample in the group. It's creating the batch report. It's actually creating a whole file folder full of png images. Then it's going to open those images up in my web browser and display them one by one.
Here's my report. Here is the original layout that I was doing and here is the report in a web browser format and I can scroll through and I can see the responses. Anything that I setup to display here can be batched across all the samples in your groups and then you can review it in an external environment or send that report to somebody else in your group, in your research group to review.
I like doing PDFs as well. If I want to send something it will show me ... It can focus this on any group so in this case I'll focus just on the all stain samples that only have the 20 all stain samples in it and create the batch report and it goes through the process of pushing out these images in a PDF format where it'll show one sample per page with this information. When it's done I can setup so it automatically opens up my default PDF you run I can go ahead and review all of those samples. Okay?
These is just one way, we're working on a workflow here where we load samples, create some groups to parse them out, go to the graph window and create a gating hierarchy that you think is appropriate but you usually are only creating that gating hierarchy on the single sample to start. Then I go and group apply the gating structure to all the samples that I need it on, make a profile view of the responses and the gates that I created for my assay here and then batch report to some type of report that I can review. Again, you can review here in FlowJo and the purpose there of just scrolling through the samples one by one is you want to make sure each of the gates that you created is appropriate for every sample.
If you need to make modifications here you can just go to ... You can highlight the graph plot that you don't like the gating for, right click, open the original graph here, there's a right click option it'll open the graph window where I can then move or move or change that gate in any way I want. When I move that gate it actually grays out that gated population on that sample. I'm on my LD14 no stem sample right here and there's the gate. It's now gray. It's not red which denotes that it's different from all of the other group owned gates. If I want to apply that new change that I've made I just highlight the gray gate, right click and copy that analysis to the group and it'll override the existing gray gate. Now the change I've made has been updated to all of the samples.
I'll also mention that at the level of a group you can use the synchronize checkbox to turn on synchronized group owned gates so that when I make the change on a single sample it will automatically update and I don't have to reapply my change to the gro owned gating tree. You just want everything to be the same. You can synchronize them and then when I make one move the gate never turns gray because it's updating automatically. Okay? I'd have to apply the change. Then when I change it all of my stats update downstream for everything. It's an iterative process, I'm usually going to go through and take a look in the layout editor for any bad gates on a single sample and make my modifications, apply those to the group gating tree until I have a gating structure that works for all the samples and then generate a report for posterity and give that to a manager or a colleague to look at.
The better you can illustrate your analysis and share your gating approach with you colleagues, the better off your gating will be. More minds are better than one. This is a great place to stop and say "Hey, what do you think of my gating structure?" to somebody in the lab that knows more about this than you do or talk to your boss about what they would like to see in terms of the analysis approach. There are once you have all of the numbers that you want to get out, and I'll show you that, then we'll want to make a layout or table and actually generate a report of each of these samples with all the numbers of my analysis.