1:33
One is the height.
So that's the you know, the,
the peak height of this, of this curve, and the other is the width
of this curve defined at a particular percentage of the height.
And the third is the area.
So, if you were to shade in everything under this curve,
that would be the area of the event.
So, you can quantify area, width and height.
And, very often people like to set a threshold for an event.
So, they, they won't just collect every peak.
They'll, they'll say, well only if the signal
crosses above a certain threshold, are we going to consider this a real event.
2:18
So the first thing that people usually look at in a flow cytometry experiment is
forward versus side scatter, and this can actually tell you quite a lot about
about your sample, and they look at this in a so
called 2D scatter plot which is just an X-Y plot of, of one versus the other.
And this is what it might look like if you had a a sample of blood, and
just based on the light scattering properties of this sample.
So if you plot side scatter here on the y axis versus forward scatter on the x axis,
and each dot corresponds to an event.
5:09
So when you incubate a,
a population of cells with this Annexin-V labeled with a fluorophore.
It will only very strongly bind to cells where this phosphatidylserine is
exposed on the outside.
Meaning that there's,
there's something going on in this cell that's, that is not healthy.
Meaning that it's it's not turning this phosphatidylserine back
into the inner-leaflet of the cell.
So, you can see in this case we took a population of cells and
treated them with a factor called TRAIL which induces apoptosis, this is actually,
a reasonably large dose here.
And if you look at what happens over time,
5:49
with a dot plot versus the viability stain, Draq7 and Anexin V.
You can see here a short time after the TRAIL treatment,
most of the cells are viable and they're not having Annexin-V staining.
But as time goes on, you have more and
more cells that are starting to show Annexin-V staining.
So, here after ten hours, you have slightly more.
Here after, after almost a day you have quite, quite a bit more, and
also you're seeing a shift up here to unviable cells.
So, the cells aren't on, only undergoing apoptosis but
then they're essentially dying.
And then becoming permeate to this viability stain.
Another very common application of flow cytometry is cell cycle analysis,
so you can look very rapidly at the state of the cell
cycle across many cells in a population, and you do this by using
a DNA stain typically one that's used again is propidium iodide.
So, not only is propidium iodide a viability stain it
also binds very strongly to DNA.
And, the difference here in cell cycle analysis is that
it requires cell permeablization and cell fixation.
So, what, what that means is that you collect your sample of cells, and
instead of leaving them alive, you use,
something which, freezes them in their current state.
It's called a fixative.
A common fixative is, for example, formaldehyde.
8:23
and, so I'll just note here that propidium iodide actually binds to RNA as well so
you have to do things which I like to treat with RNase that
degrades all the RNA in the cell.
the, the prebic predominant amount of RNA in the cell is actually ribosomal RNA, so
you degrade all that.
And then you can get good good data which
is reporting only on the levels of DNA in the cell.
So this is the type of data that you would expect from a DNA staining experiment.
If you look at histograms of the number of events, or number of cells that have
a particular amount of fluorescence intensity of your DNA stains.
So for example propidium iodide,.
Cells that are, that are down here are so-called G0/G1 having you know, i,
9:56
You can combine these DNA dyes with different types of
staining which tell you, which give you additional information about S phase.
So for example you might have a treatment condition which
halts cells in S phase, so.
Cells might have S phase DNA content, but
they might have stopped cycling due to one reason or another.
So you can assay that by doing a pulse of something, which can be detected,
incorporated into the DNA of cells that are actively replicating.
10:44
so, in this case you know,
again, a FITC labeled antibody is a very common one that, that can be used.
And you can, you can take this DNA staining information and
then combine it whether cells are positive or negative for BrdU incorporation.
So an example here that then you can get this information of G0, G1 and G2-M.
But you also know whether these cells in between the, they kind of two copies and
four copies of the genome are actively replicating their DNA or not.
Another very very common application of flow cytometry is in immunology.
For immunophenotypings.
So, the ways that, that you can tell different types of either lymphoid or
erythroid cells from one another is by surface markers.
So you can take a sample of blood or some other type of immune
system relevant sample and you can incubate them with antibodies that reckon,
that are specific for these different extracellular markers on the surface of
cells that are conjugated to different flora forms and.
By doing these sorts of quite simple experiments you can start to, to determine
what are the relative frequencies of the different types of cells in your sample.
So just, an example of one map here how, you know, you can go from your
hemiparetic stem cells which are known to express certain types of markers here.
Various types of you, you'll see lots of these markers that are just C-D and
then a number these are just different receptors that are expressed as,
as as these cells mature along different paths of differentiation, so
you can tell you know, is are these cells progressing into the lymphoid line.
Are they are, are they turning into T cells, are they going,
turning into natural killer cells, are they turning into B cells et cetera,
just by the pattern of standing of these different extra cellular markers and
a very quick flow cytometry experiment you can tell a lot about the immune system.
Okay, and of course as I mentioned before in DNA cell cycle
analysis you aren't limited to looking at things that are on the outside of cells.
You can also look at things that are on the inside of the cells,
as long as you fix the cells and then permeabilize the cells.
In the case of cell cycle analysis usually in ethanol or
alcohol fixation, it is sufficient.
Because the DNA is really contained in the nucleus and
it's not going to so called leak out of the cell.
But in other cases, say you're interested in the levels of a protein or
the levels of phosphorylation of a protein you can look at that via
flow cytometry but if you permeab, permeabilize the cell before you fix,
a lot of these proteins may leak out or be lost.
So typically in this case you use a much stronger fixative, such as formaldehyde,
to, to really cross-link everything that's in these cells, and
then you can permeabilize with an alcohol, such as ethanol or methanol.
Which allows you then to get your pro fluorescent probes into
cells in this case, you're looking at some data where they used,
antibodies against specific targets, which were coupled in different floor fours,
14:50
And then, you can look at how the levels of activated are, depend on the expression
level of the various proteins which you've stained for in the population.
So you can do this kind of multicolor single cell experiments and
really look at dependencies in that kind of a way.
Okay, I doubt there and, another pretty fascinating thing you can
do with flow cytometry is so called fluorescence bar coding.
So you've all probably been to a, to a retail store and you know,
seen a lot of, many of the items which you buy from a rea,
retail store are coded by a series of white and
black lines that are kind of arranged parallel to each other.
And the thickness and
spacing between these lines, it contains a lot of information.
It's unique.
16:29
You wash it and make sure that the fluorescence bar code is, is like,
nice and unique and clean, then you can combine those samples into one tube
because the cells from each tube have now, now have a unique bar code which you can,
which you can figure out in the flow cytometer.
And that allows you then to, instead of running many,
many samples in the flow cytometer, you're only running one sample.
Which really makes the experiment a lot easier.
16:59
And it also reduces many forms of variability associated with
the subsequent staining for
singular epitopes the variability on the cytometer from sample to sample et cetera.
So, an example of how this works.
so, let's say you had three fluorophors here that you
were using to define your fluorescence bar code.
And each one of these fluorophors you could add in at different concentrations.
So you can have none of it, you could have an intermediate level of it, or
you can have a very high level of it.
So you have three levels times three different fluorophores gives you
the ability to define 27 different samples based on fluorescence bar codes.
So, for example if, if you look at, you know, what would a series of events with
this first fluorescent bar code look like, you would have you know, if you look at
the, intensity of that first flourophore versus some other parameter.
In this case, side scatter.
You would see these three different lines of
events corresponding to how you have bar coded those cells.
Okay?
So in this case there's this highest population here,
this highest row would correspond to this row C here where you put in the highest
concentration of your first fluorphor this DyLight 350.
Now if you then so called only select those cells in this row,
something called gating which I'll explain a little bit more later.