Okay. So that was a little bit about how a flow cytometer actually works, and you know, what are the characteristics in cells that you can, that you can measure. But how do you use a flow somet, flow cytometer to measure something relevant? You know, what are the types of experiments that you can do? so, the first concept you really need to understand in order to, to appreciate that is the concept of an event. And an event is defined as whenever some object goes by the laser, coming out of the flow cell and going by the laser. You can imagine then temporally, because it's a flow-based system, that the, the object, which is usually a cell, doesn't always have to be a cell. Sometimes it's, it's so-called debris or junk. You start to get a signal. Say for example, this could be the forward scatter channel. You start to get a signal which then as, as the center of this object passes the laser you get a, a so called peak. And then, as the object flows out of the laser path, the signal goes back down to zero or background. So there's several features of this event curve which are quantified in a flow cytometry experiment. 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. 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. You know, essentially a cell that has gone by the laser and then these light scattering properties have been collected for that cell, or event. So cells up here on the four scatter side, side scatter space are typically neutrophils of this particular properties. You can identify monocytes or lymphocytes so you can identify different types of cells just by looking at the different light scattering properties and you know, this is true of many different types of samples which you might have. Another thing that one can measure is viability in apoptosis. So these are, these are some data over here where, where we used a couple of different stains. One is called draq7 but there's, there's many stains which are fluorescent dyes, which are cell impermeant, meaning they don't get into the cells. Unless the cell membrane is compromised, indicating that the cell is just unbiased, so called unviable or dead. Propidium iodide is another one that's used for this. So if you incubate yourselves with this viability stain, the cells which are positive for the stain will be so called unviable. And you can collect this data then, on a log scale, here, as I'm showing here. And another stain can be combined with is a molecule called an Annexin-V which is conjugated to a fluorophore. often, it's conjugated to FITC, the one that I, that I described before. Which is spectrally compatible with this molecule Draq7 which is a far red fluorescence. And a FITC of course is a green fluorescence as as I described before. Now Annexin-V will only bind what binds very strongly to a molecule called phosphatidylserine. And as long as the cell was alive and healthy, there are enzymes that, that keep phosphatidylserine on, only on the inner side of the cell membrane. 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, 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. In this case you use a, alcohol fixative, usually ethanol. And sometimes the fixative will permeabilize the cell, meaning that it, it makes the cell membrane be in such a state that now, fluorescent dyes or labels can get into the cell. The ethanol seems to do actually a reasonable job of both fixation and permeabilization, but not all fixatives do that. But regardless that's the difference between using a dye like propidium iodide for viability. If the cells aren't fixed and permeablized it can be used to report on viability, but if the cells are fixed and permeablized it can be used to report on the status of the cell cycle because it binds to DNA proportionately, stoichiometrically. And therefore the fluorescence emission of propidium iodide is proportional to the amount of DNA content that was in that cell. 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, in the case of a diploid genome two copies of, of of all the chromosomes. If cells are in S phase, typically they have between one and two copies of the genome here. And if cells are in G2 or mitosis phase, then they have four copies of the genome. And so they have doubled, essentially doubled the fluorescence intensity of those in G0G1. So, by looking at this distribution as a function of how you might treat the cells, you can get an idea of what's going on in the cell cycle, in response to your treatments. 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. And then can be detected later. So one of these molecules is called Bromo, Bromodeoxyuridine or BrdU. So, this gets incorporated into DNA by DNA polymerase during DNA replication. But then it can be detected by an antibody. 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, actually a very nice paper here which I won't go into detail on rather to say as an example of how you can use flow cytometry to look at the levels of, of different proteins or epitopes within cells. On a single cell level in a population. And then look at how they depend on one another. So in this case, they were looking at the levels of doubly phosphorylated erk. It's a it's a particular kinase, which is implicated in the control of many physiological processes in mammalian systems. It's actually conserved from yeast up to mammals. This particular map kinase molecule. 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. And this allows you to say well, this bar code stands for milk and this bar code is for orange juice. Or this bar code stands for eggs, et cetera. So you can do a similar type of thing in flow cytometry. Except instead of using series of black, black and white lines, you use different patterns and intensities of fluorophors. This is so-called fluorescence bar coding. So I'll explain it a little bit more on the next slide. But the idea is that it allows you to much more easily deal with a lot of treatment conditions at the same time. So if you have lots of different treatment conditions you can take the cells from each treatment condition which are on different tubes put a different fluorescence bar code in that sample. 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. 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. Then you can start to look at what's happening with your second fluorphor. So, I'm only selecting these, now I'm saying, now what's happening with my pacific orange fluorescence part of the bar code. And again, you see the three different combinations depending on whether you put nothing, a little bit or a lot. So again, now if you look at this case where you put a lot, gate, now we're gating only on these ones, and only on these ones. Then you look at your final bar code, you again see your three three rows corresponding to unique bar codes. So, by deconvolving and gating in this way you can really increase your ability to multiplex in flow cytometry. It's a very powerful technique. Okay. So, the next series of lectures that I'll be giving are on mass cytometry. And that will be in the next set of lecture slides.