This content provides a foundational tutorial on basic operations within the FlowJo software, guiding users through essential steps from data import to analysis and presentation for flow cytometry data.
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Hi and welcome back to the channel! I've had a number of requests come in for
FlowJo help and so today we're going to look at some basic operations in FlowJo. So
instead of looking at my face you'll be looking at my screen. Basic FlowJo operation number one:
getting your files into FlowJo. The easiest way I found is simply to drag
and drop. So you'll see in here I now have my FCS files have populated into my FlowJo workspace.
Now on the side here you'll see three toggle buttons. So the first one denotes which sample
you're looking at. So you'll see when I click on the sample and the plot opens up
that diamond gets highlighted. The circle looks at the data quality- so this is something you can
find under tools and check sample quality. It will run through and look at the stability of
your fluidics in these samples. So if you click on that you can see my sample quality was okay
and my file status is okay. And when you open it it will give you an overview of your parameters
over time. So it's a nice way to screen through to make sure you had good collection of the data.
Then in this next box that is where you'd find your compensation matrix.
Now in this experiment, we only have one color so there is no compensation applied.
We will look at the compensation and how to do compensation in a subsequent video.
So from here, the next step you would go into would be gating your samples. So in this case
I would first want to start by gating around my cells. So drawing a gate,
every time I click I get a new point. I like the polygon gate. You can play around with the
circle gate or the rectangle gate as you see fit. And then you can give that population a name now.
When you double click inside of that gate it will automatically start a hierarchy. So building down
from gate to gate and in here if we switch this to our forward scatter area versus height you can see
we have our single cells and then our doublets. So we can use this to get rid of those doublets. And
as soon as this is done you'll see I now have a single cells gate underneath my cells gate. So we
have established our gating hierarchy. Now these numbers sometimes end up in really odd places
so you can grab them and move them so that they're not sitting right in the middle of your population
and put them wherever you like to see them. So double click again and we'll start looking at our
TLR4 Alexa 488 stain. Now you'll notice we have a lot of white space on the side here which makes
our background population then look a little bit funny so to adjust this we can click on this T
and go into customize axis. And in here the easiest thing to do is just change the width
basis. So this changes how much of how much space there is for the bi-exponential area of the plot.
So by decreasing this you'll see that area will get removed and our background will spread out
and that gives us a more normal looking background. So from this one here we can set
our gate because we know where our background is so this is where we would find our TLR4
positive cells
and again adjust that and now that we have our basic gating hierarchy set up. We want to apply
this gating strategy to our group- so there's two easy ways to do this. First off you can drag and
drop and apply that to all your samples. You can also highlight these gates right click and
copy analysis to group. End up in the same space either way so choose whichever one works for you.
So now that we have our basic gating hierarchy set up, the next step is to look how our gate
has applied to our population. So to do this we can open up a plot and scroll through to see how
this gate looks on our various samples and you can see that the gate placement appears to be correct.
Our secondary alone shows very little staining which is both great for our gate placement
and for our protocol and we have decent staining with all of the concentrations used. After this
we would then want to go on and present our data. So we're going to do this with the layout editor.
So on here you're going to make the collection of plots that you want to show to present your data
to someone else. So to do this we're going to drag and drop various populations onto our workspace.
Now one thing I will note is a lot of the times the gating hierarchy can get a bit confusing with
the plots that you want to see on here. So if I want to look at my TLR-4 positive cells
if I drag this gate on I'm going to see the cells inside of that gate. So if I want to see-
I'm sorry wrong button undo that- so if I want to see where my gate lies to show my TLR-4 positive
cells, I actually want to go a level above and grab that plot. So you can see here by looking
at the single cells where I have drawn that TLR4 positive gate I can now see that gate applied
to the population. Whereas when I grab this plot I only see the cells inside of that gate.
So I would start first by bringing in my forward scatter side scatter plot.
And one nice thing about the FlowJo layout editor is you can adjust the page size to
whatever you want to be simply by grabbing in the middle of the dotted lines and drag it around. So
if I want to make this a bit more of a larger layout view I can do that so from there. I'll
move down my hierarchy bring in my single cells gate and then bring in my TLR4 gate
now. If you've ever trained with me before I have what I like to call flow plot OCD-
I like all my plots to be lined up and all of them the same size.
So over here in the arrange button there's some nice features where you can do that. So you can
arrange everything to the top, you can space them so that everything is nice and linearly laid out.
Some people also like to use this line feature to draw lines from so this gate goes to this
plot. Again you can do that here or you can do it wherever else you want to analyze your data.
So once you're on here and you have all of your plots laid out that you want to look at
the easiest thing to do is create a batch report. Now I like to use this little
separate pages button because then I know that every single sample that I have in my
experiment will be on a separate page in my layout .So it'll be easy for me to work with afterwards.
And then click this create batch report and then I will end up with this lovely report
that has the gating strategy for every single one of my samples. Now I realize that this is a very
simple straightforward experiment however these steps don't change no matter how many plots or
gates you have. You simply will have more things in your layout. So now that that's saved, you can
go ahead and export it under the file tab here and export image in whatever format you'd like and it
will kick out your batch report. So that is now over here on our desktop. So if we open this up
you can see we now have our batch data file with each page being one of our experimental samples.
From many of your experiments you will want not only the flow plots off of your layout editor but
also a data table that has the statistical plots that you have in here. So this is done in the
table editor and is it as easy as going into edit, adding column, and then choosing the statistic
you want to see. So in this case if I want the frequency of my TLR4 positive population I'll go
frequency of parent of TLR4 positive and ok. And then that will give me from every single sample
the percentage of TLR4 positive cells. Now in here if you want to give this a gate name you can
or you can simply use whatever is in this will be the default name when your table is created.
Another commonly used one would be median. So if we want to look at median in say the single cells
of our TLR4 stain, we can add that in as well. So from here we can then go ahead and create table.
Now there are some nice options here where you can either do it to display- which is what I'll
do today so it just shows up. But you can also copy it to a file to your current layout or to
a clipboard if you want to copy and paste it into another downstream program such as excel.
So for right now we'll just copy and create to display. So on here you can see we have a
table with our percent TLR4 positive gated and up here our column title is the name that we put in.
And then on the side here you can see there's a lot more in this so this is because here we use
the default name. So we know it came from our cells and single cells gate. We are looking at
the median fluorescence intensity in this channel. So you get all of that information in your title.
And then down here you will have your median fluorescence intensity for each of your samples.
So that is just a really quick overview of some basic operations in FlowJo to get you
started. The really critical things you need to know- how to bring samples in,
how to get your samples, how to get your data out, and again while this was a very simple experiment
that workflow doesn't change no matter how complicated your experiment gets.
With that being said I will have some other videos coming out looking at other FlowJo
operations. However I would like to know what you would like to see so in the comments below
please let me know the kind of things you would like to see analyzed in FlowJo and I
will work to get you videos on those as well. And with that we'll see you in the next one!
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