Immunology Fall 2024: Lecture 8 Flow Cytometry

Large but we are going to be talking about doing flowetry so much this semester um flowetry is like one of those things that immunologists read about think about do all the time and so I need to sort of give you the background on photometry and how it works um the plan for next week is that we're going to do some cytometry once you know how it works um so anyway I need to like Tell you the update of how I need we need to go through house citometry Works um like I said the plan for

next week is that we will actually um prep some samples and do some citometry um provided that my machine is working by then I've been working on it a lot maybe if I maybe I should just try to build a radio and then um I can go from there uh so that's going to be the plan for next week but we need to know kind of how the technique works because it's Going to start showing up soon in lectures and papers and all the time so we got to know what's up um the after I

kind of go through um this information um I do have a handout for you um the handout has two parts um it has some questions and it's due in two weeks um in lab the first part is honestly some basic kind of data analysis using what I'm telling you I'm showing you data from papers and asking You questions about it things like that the second part is asking you some questions about your data from from next week's lab so it's sort of an allog together flowmetry thing so there's a part one and a part two

in the handout I've got print outs of it here that I can give you when I'm done um if you want to sit here because I am not going to be talking for three hours um if you want to sit here and work on those problems and ask questions about at Least the part one questions after I'm done totally fine um but the whole packet in general isn't due for two weeks because you don't have the data yet for the other part um so uh we're going to be talking about um I'm going to talk

about some general antibody techniques um and then use that to segue into flow cytometry um so I like this in this part of the class for two reasons one because we're talking About antibodies in class and so thinking about techniques that involve antibodies make sense um but also because when we start actually talking more about B cell biology um in the future um a lot of parts of bell biology are things that I need full cytometry to explain to you fully um so all of these techniques include antibodies yay here's my antibody um and there

are Tons of techniques that use antibodies that are out there um and so you can see this table that lists some of the techniques that are out there that use antibodies um so um there's one that's listed here is precipitation reaction in fluids um I've got precipitation in gels I've got a glutin um I am now remembering that I used to Do this in a slightly different way um so I may have to I may draw some Stu on the board um but really with all of these we're using the ability of an antibody to

specifically bind an antigen and the difference with a lot of these assays is in terms of um the sensitivity so how much antigen do you need before you get a positive result and what kind of format is your sample in so do you have a liquid do you Have a gel do you have a solid do you have a cell like that's really where how these are varying a lot um just to give you some examples um you guys have um talked about Western blots in BIO 250 where you've used an you've taken protein samples

you've separated them on a gel and then you've used an antibody to find a protein of Interest out of all of the proteins that were in the whole Cell um and so in that case I have such pretty slides of this that apparently I didn't feel the need to put in this presentation um I have a tube it's full of a bunch of different proteins shown here as different little shapes I can run that protein sample on a gel and I'm going to get a ton of proteins of all different sizes and then if I

want to find my protein of Interest I can take an antibody bind it to all these proteins and detect where the antibody was bound and I'm specifically looking if this tail region um to do my detection um so you guys have seen a western block before sometimes we can do so this of course requires you to have a protein sample you can run on a gel you could also have samples in Different formats so there's one that we have where we have a sample called an Eliza where we just use a little well of a

plate and we put the sample in The Well of the plate and then we again bind our antibodies to it and see will the antibody bind or not um and so it's conceptually the same kind of thing um if you are uh need to take a Atome covid test SARS K2 test it's just it's an antibody essay it's it's a I guess I would call it a it's sort of a precipitation reaction no it's not in a fluid um where basically you have one area right here for you have a filter paper and you have

these two antibodies attached to the filter paper um and oversimplifying you have You have a thing that makes some color if it binds to an antibody on the other end of the filter paper so imagine like a coffee filter or just a dry piece of paper you put some stuff on one side you put some stuff on the other side then you swab your nose and put some liquid and you touch that right here now you made the paper wet and if the paper gets wet the liquid flows all the way across And all this

stuff mixes together and if there was SARS kv2 now the SARS K2 would bind here and it would also bind to the color thing and you get a color all of a sudden where there used to not be one on your filter paper and this one is a control that basically binds to anything and so you always get an A Line there to tell you that the test worked and so basically what you're doing is you're just allowing capillary action to flow Some liquid maybe with SARS cov2 maybe not down a piece of filter paper

it's it's again just an antibody um assay doing an antibody interaction um this exact same um phenomenon that I'm showing you here is also how a pregnancy test Works um you are putting urine that might contain a particular hormone right here it's now flowing down this thing through capillary action and either binding to an antibody for the hormone Or binding to a control antibody they're fancy they make it so the lines are perpendicular to each other so it becomes a plus or a minus but it's that and if you think about all of your covid

tests where you're waiting for the two lines that's all this is all you're doing and so all of these things are just ways we use antibodies and we can use them in so many ways for so many different biochemical assays yeah M so the O the antigen is only in the test if it was in your nose because you're gonna swab your nose and put it on there with some saline and so if you have the virus then now there is antigen um if you don't have the virus then there's no antigen either there is

something that binds a control either way there's an antigen that binds at control but in terms of the SARS CO2 it's a question of was there SARS K2 in your Nose no it's detecting whether the antigen is there to bind to the antibody that is on the paper yeah I know like you can use that for a strut test alternative swab they go usually if they're doing a swab they then culture um so not an antibody test in that case so so many things out there are really taking advantage of antibodies and their specificity um

for the technique that we're going to talk about there we are going to be trying to answer kind of a Specific question and we're going to be doing it using antibody specificity and this is a question that you guys have already thought about in lab one um so in lab one remember you had to tell the difference between all of these cells right by I um and it can be hard to tell the difference between them by ey and sometimes you get things like a Lymphocytes there's B cells and te- cells there's actually kinds of

B cells and te cells I can't tell you if this is a B cell or a T Cell all I know is it's a lymy um so we have some sort of limitations from this process that we talked about um and there the that's sort of not the only sort of limitation for this but um just To well well okay one limitation is that if I actually looked at blood white blood cells would be really rare so I'm looking for needles and Hast Stacks if I want to do it if I want to take some

blood and study lucaites and start to study all sorts of different cells it's a needle in haast stack you guys all came up with a percent of what percent of the um blood was lucaites and you all direct dramatically overestimated because you all picked up A a field of view that had at least one in it so you all got 1% and it's way less than 1% um in reality um so a we're trying to look for needles and Haystacks which is pretty hard um we also have this problem where some of these cells that

you saw here were are are really heterogeneous so this uh these charts uh that both this this chart here and this Pie chart show you some of the kinds of lymphocytes that exist and the point of the story is when you were doing that count in lab one you were like lymy and you did not do things like try to figure out which of these many kinds of lympocytes um you had but there are lots and lots and lots of situations where we need to have that kind of Information um so you guys in your

uh answers to the lab one questions gave me some suggestions of how you could make your data better how you could kind of deal with some of the difficulties here and what kind of things did you come up with of things that could help you deal with the difficulties yeah Jonah okay so if we had a way to remove red blood cells then that could make our Life a little easier sure yep we could use a larger sample so what's the deal so like what would a larger sample help us with h so you could

get a lot more accurate if you weren't doing a 100 cells If instead you tried to do 100,00 cells right how would you feel if I asked you to repeat lab one but tell me 100,000 cells you're all making some excellent Faces about that so what what's the problem with that scheme of doing a 100,000 cells yeah just too tedious it's tedious it would take forever right it would be not fun the other thing is that so we I'll come back to that the other thing that we could do is have some way that we

could get more information than this about the cell if we could somehow get more information than we're getting here that would Either make you more certain or it might help you like determine is that lymphocytes so how long did it take you to classify a 100 lucaites in lab one yeah took J our 15 minutes okay 15 minutes for a 100 okay anybody else yeah 16 yeah 25 minute no all all good all good so you see kind of what we're talking about this technique that I'm going to introduce you to today um when I

was a graduate student every Experiment that I did um had a minimum of 100,000 cells um and every experiment uh took a Max of two minutes so I was getting 100,000 cells in two minutes no that's just to put it in the machine the prep took a little longer than that but the machine would actually analyze 100,000 cells in two minutes in fact I can get you can get it to go higher um to go faster than that so what I hope you can notice here is exactly How powerful this is and how useful this

is um in doing this analysis that um we're thinking about and this is the type of question that I was trying to address as a grad student so I was actually infecting mice with some viruses and I was measuring the number of cd8 cells that responded to that virus over time to look at primary and secondary responses so I have graphs that I publish that look approximately Like this so I was measuring that virus specific T Cell CD T cell and making a graph like this okay that's I legit have published graphs like that what

that means is I had to have a way that I could tell the difference between a cdat cell that bound to HIV and a cdat cell that bound to a different antigen like a flu antigen and not only did I have have the ability to do that even if I did as Jonah said which I did and got rid of all the red blood cells um I actually had to be able to pick out that cd8 cell that responds to HIV from all of the other white blood cells in my mouse blood and so and

I look at this slide and I think about me when I started teaching because I actually spent the time to like make the number of cells correctly proportionate on this slide and now I like I so would not do that right now But that's okay um so you can see like needle in a hay stack but yeah if you look at a lot of Immunology papers this is sort of very common a very common kind of data that we might have and so the way that we deal with this is that we know that lucaites

have characteristic proteins on their cell surface so if I think about all of the types of cells that you guys were looking at under a Microscope I can tell you that they have SE uh proteins that we've described that we know of on their cell surface I'm going to tell to say examples right now you don't have to like have these examples memorized for later as we talk about different cells throughout the semester we'll say what the ones are for that cell but I I it's easier if I give you some examples right now so

for example a t- cell has a te- cell receptor it also has a protein Called CD3 and immunologists just know that and it also has a protein called cd8 if it's a cd8 T Cell It's a CD4 t- cell well it still has CD3 because it's a T Cell but it has CD4 if something's a neutr it has a special protein called l6g and so there's like a pattern for all of these cell types of proteins that are on their surface um over the years immunologists have spent a bunch of time cataloging the proteins that

are on the Surface of different lucases and so you might imagine that you all took your favorite Lucas site and found the proteins on its surface or started finding proteins on its surface and so let's imagine that Brena what's your favorite lucite I don't know what's your favorite that's a really hard Question does anyone anyone have a favorite Lucas site I need at least two people with favorite lucases for my example a natural killer cell all right Brian really likes a natural killer cell so let's imagine that Brian and Josh both like really like natural

killer cells okay and they start describing proteins on the surface of the natural killer cell Brian you just found a cool protein On the surface of natural killer saw what would you like to name it h you can name whatever you want right the Josh and Brian protein now Josh you have a separate lab you have just found a protein on the surface of a natural calor cell what's the name of your protein nk16 all right so we got nk16 and we got the Josh and Brian protein here's the question is nk16 and the Josh

are are those the same protein with two Different names or are they one protein or are they two different proteins you don't know right you you it sort of becomes a little bit tricky because we don't have a standardized way of doing this process a standardized way of naming for a while we didn't have a standardized way of of naming it led to papers like this I had to read this paper as a grad student they had to list every name that the protein had ever been given so in This case the CC chemokine thymus

derived chemotactic agent four there's one name um secondary lymphoid tissue Kine there's a second name six cine there's a third name Exodus 2 is a fourth name so they had to list all the names for this protein because it didn't necessarily have a standardized way of naming it that seems lame right and it also seems like if you were a researcher so now Jonah also loves NK cells and Jonah Decides he's going to start doing some experiments on enk cells he reads Brian's paper he reads Josh's paper he tries to figure out his experiments and

he's like I don't know if these are the same proteins or not and I don't know what to do and I'm confused and it's annoying right it's all very annoying so what immunologist did is they came up with a standardized naming system for these proteins there is actually a group who Gets together once a year to look at all the proteins people have found on the surface of of lymphocytes and give them standard names and figure out like which ones are all the same and which ones are different you can tell the people who are

part of this committee must be really fun at parties um if that's what they really want to spend their time doing but whatever um and this is called the CD System um and so the CD was originally uh named as cluster of differentiation because people thought something might be going on with different differentiation States no one ever says cluster of differentiation anymore just go with CD but basically we just made a list so we said aha we found a first Protein that's on a Lucas site we're going to call it cd1 and then somebody else

found one so so Brian's protein now gets called cd1 And if we figure out that Josh's protein is different than Brian's we call it cd2 and if we figure out the Jas is different we call it CD3 and so on we're up to about CD 400 um in for most immunologist right now the back of most Immunology textbooks have a CD chart um in my lab I have a like I have cd charts in my office do you need to have the CD charts memorized no I'm going to tell you about the important CD Molecules

that are important for a particular cell type as we get to that cell type but one of the things people are always when people make fun of Immunology they're like oh my God those immunologists they love their their CD jargon it's just putting a number on things so things aren't Annoying It's not like trying to be all impenetrable it's just that we're trying to be a little bit organized and so we have cd1 cd2 cd blah blah blah blah blah blah Blah blah blah so one of the ways that we can address this needle in

a hay stack problem is we know some of these characteristic proteins on the surface of a lot of these cells and they have these CD names in most cases you can see there are some that don't have a CD name listed there um but that's because the old name stuck they actually do have cd names too um so this so we can actually look at these Proteins on the surface of cells to help us know which cells are which and you might be like awesome that's great Dr Barker um how do you look at a

cell and know what that it has a protein called cd8 on its surface those glasses you're wearing must be extra special if you can actually see cd8 proteins on the surface of a cell and the way that we actually really do this Maybe I should push the wrong thing right thing let's use antibodies so we have antibodies against all the CD markers and so we'll take our cells of interest and we will add antibodies to them and usually those antibodies have a modification where they're going to be fluorescent so for example if I wanted to

detect a cd8 positive t- cell I know that T cells all have CD3 on them cd8 T cells have cd8 and so I could use an Antibody against CD3 that is blue it has a blue marker on it and that's going to find all the t- cells going to turn all the t- cells blue and I could use it an antibody against cd8 that's going to turn all the ones with cd8 uh red and so I can look and see well how many cells are both blue and red and those are going to be my

cdat cells is basically how this is going to work um does that part make sense do we Get what's going on here yeah okay so that's all cool um this is a big part of what we're doing but there's still one other piece to this because you might still look at this and be like awesome Dr burer now we're going to look at these um under a microscope and we're going to instead of looking for the things we were looking for before Look for Blues and reds do you think that that is going To speed

you up very much compared to what you did before no so it's a way you can get more information about the cells yay but it's not going to help you with your speed problem if really all you're doing is hey let's look at a let's look at cells under a microscope and see which color they are that's not helping you with the speed at all it's going to be just as long and just as annoying even though Yes you're getting more information so the other useful part of this is that we have a machine that

speeds this whole business up um and that machine is called a flomer um and so here is um a little a little bit of info about the flomer um I'm going to explain it and then I'm going to show you a little video um that talks through it so what we do is we first take our cells and like do some pipetting and Stuff with them we do some we prepare the cells so up here you've got you know your sample which is your cells that you've prepared what you might have done in preparing those

cells is you might have added antibodies of Interest so you like took the spleen cells from your mouth and you said I want to know about cd8 T cells in my mouse so I'm going to add my anti-cd3 and my anti- cd8 antibodies so I'm going to like mix it all together And you could see that if I did that some cells would have no antibodies bound to them some cells might have one some cells might have multiple all depending on different proteins that are on the surface of those cells and I'm going to mix

my sample with a huge excess of liquid notice that my sample up here is a heterogeneous sample so it's not like I purified the sample at the beginning it's just the whole mixture of all the Cells that were in the blood or spleen or the whatever um mixed together um so again I'm still I I basically just threw a whole Hy stack in the machine and I'm hoping that the machine is going to tell me where the needle is and what the machine is actually able to do is use um changes in pressure and other

electrodynamics to make the cell to make the cells fall in droplets of one cell Per droplet and so the machine is going to take this whole mixture I'm going to like type on my computer a little bit and I'm eventually going to get the machine to drop the cells you can see the sort of cells dropping right here one cell per drop are just going to get dropped through this area so here you can see the stream of fluid drops containing cells some drops might contain this cell That had no antibodies on it some drops

might contain a cell that had one antibody on it some drops might contain a cell that had multiple antibodies on it um and this stream of drop is then going to be hit by a laser so it's basically going to go into a laser and the way that the cell and any antibodies attached to it inact with the laser light will be detected on a computer at the End and so I'm going to then take the information that comes from that computer and I'm going to use it to um give me information about the cells

and again this is where I can set it up so the speed of those droplets is letting me get like 100,000 in two minutes because it's going to go really fast and just shoot them with the laser the computer's going to read out information about how that cell interacted with the laser light we're Going to move forward so that's the sort of broad overview of cytometry see if this actually works flowetry is a powerful technique for the analysis of multiple parameters of individual cells within heterogeneous populations flow cyers are used in a range of applications

from imot to analysis to cell counting and gfp expression analysis the flowy performs this analysis by passing thousands of Cells per second through a laser beam and in the light that emerges from each cell as it passes through the data can be analyed statistically by flowetry software to report cellular characteristics such as size complexity phenotype and health in this tutorial we will look at how a flow cytometer Works how scattered light and fluoresence are detected by a flow cytometer and how the resulting data can be analyzed this view shows the primary Systems of the flow

cytometer schematically these are the fluidic system which presents samples to the interrogation point and takes away the waste the lasers which are the light source for scatter and fluoresence the Optics which gather and direct the light the detectors which receive the light and the electronics and the peripheral computer system which convert the signals from the detectors into digital data and perform the necessary analyses The interrogation point is the heart of the system this is where the laser and the sample intersect and the Optics collect the resulting scatter and fluorescence first let's talk about how the

sample is delivered to the laser here we see how the sample is transported through the interrogation point for accurate data collection it is important that particles or cells are passed through the laser beam one at a time most flow cytometers accomplish This by injecting the sample stream containing the cells into a flowing stream of sheet fluid or saling solution as you can see the sample stream becomes compressed to roughly one cell diameter this is called hydrodynamic focusing in fact flow cytometers can accommodate cells that span roughly three orders of magnitude in size in most cases

cytometers will be detecting cells between 1 and 15 microns in diameter although through the use of specialized Systems it is possible to detect particles outside this range um so what I hope that you sort of got from there is this idea of how we're going to put this heterogeneous sample um in um our machine and how we're then going to focus it one cell per droplet through our stream that's going to go through the laser and that we're going to get some additional information about now I'm going to try one more thing let's hope it

works now let's see How laser light is used to detect individual cells in the Stream as a cell passes through the laser it will refract or scatter light at all angles forward scatter or low angle light scatter is the amount of light that's scattered in the forward Direction I'm going to draw this on the board in a second to the magnitude of forward scatter is roughly proportional to the size of the cell and this data be used to quantify that parameter but how can we record this Scattered light light is Quantified by a detector that

converts intensity into voltage in mosters a blocking bar called an obscur is placed in front of the foror as C crosses the laser light is scattered around the obscuration bar and is collected by the detector because small cells produce a small amount of forward scatter and large cells produce a large amount of forward scatter the magnitude of the voltage pulse recorded for each cell is proportional to the Cell size if we plot a histogram of these data smaller cells appear toward the left and larger cells appear toward the right a histogram of forward scatter data

is a graphical representation of the size distribution within the population such a graph only presents one dimensional data so here's a bunch of tiny cells and here's a bunch of big cells scatter as we have already seen a cell traveling through the laser beam Will scatter light at all angles so what what this just told you about is one of the ways that the light interacts with the cell so when I am using my phytometer I am able to have to look at the way that the light interacts with the cell in in two ways

basically those two things are telling me two things about the cell itself and its physical parameters so they don't require any antibodies they don't require anything it's just that different kinds of cells Interact with the laser light in different ways and you just heard about one of the two parameters the two physical parameters that we look at that physical parameter is called forward scatter and the way I like to think about forward scatter is it's basically how much the light is bent around cell so you can see I will have might have a small cell

it has a small forward scatter if I have a big Cell it has big forward scatter and I can plot if I have a a graph and I put forward scatter on it my small cell was here my large cell was over here and I I put them both low because there was only one of each though there are different ways I could do that graph we won't get to the graphs in a second so this is sort of what we're getting with forward scatter and that's what the um video just Explained there is a

second physical parameter that we also like to look at and so when I think about forward scatter I'm thinking about size the other parameter that I often will look at is as the slide kind of already tells you um side SC and the difference with Sid scatter it's not measuring the size of the cell but it's measuring how much stuff Is inside the cell sometimes people call S scatter a measure of internal complexity or I off I was always taught it as granularity this is one where I definitely need the video to give me like

a real drawing I'm going to show you some things and it's those of you who know some about physics are going to be like yeah that doesn't make any sense at all but that's because I can't draw um but basically the idea is some cells have very few granules Don't have much stuff inside them and some cells have a lot of stuff inside them like a lot of granules remember our granula sites and if we shine light on this cell with nothing inside it it's not really going to get bent through that cell if we

shine light on this cell that has lots of things inside it it's going to get it's going to bounce around off Of all this stuff and that light's going to be different and so basically we're looking at how much does the light bounce around because of stuff inside of the the cell or how granular is the inside of that cell and so we can distinguish every cell that goes through our flow cytometer based on its size is it big or small or medium and based on its granularity does it have a lot inside or a

little inside And so this will now say about granular about side scatter if I can hit the play button light scattering at LG Ang for exle tode is caused by granularity and structural complexity inside the cell this scattered light is focused through a lens system and is collected by a separate detector usually located 90° from the Laser's path the signals collected by the side scatter detector can be plotted on one-dimensional Histograms like we saw for forward scatter onedimensional have seen so far do not necessarily show the complexity of the cell populations for example so if

this was a side scatter plot these cells have low side scatter they're like between 0o and 50 so they probably don't have very much stuff inside them these cells are kind of medium they're like between 50 and 100 so they have some and these cells are Really high and so they have a lot of stuff inside them um this is just showing it as a histogram so how many cells are in each group um and we could I could have paused the other thing about and it was doing the same thing of how many were

big how many were small um what is also really cool about this is we don't just have to look at the data in a histogram we can actually I could change this instead of having cell count on one Axis and a parameter like side scatter on one axis I can have two different parameters on the same on on the two AES so I could have forward scatter on one axis side scatter on the other axis and that if if I did that i' get a plot that looks like this um I I went to grad

school on the East Coast so I draw my plot this way I have since learned that people who will go to grad School in California draw their plot differently but that's fine This is how the plot works because I say so um and so I'm going to you know we'll say I might look at a 100,000 cells and the first cell might be kind of tiny and not have any granules and so it would be low forward scatter and low side scatter and then I might have another cell that was huge and had tons of

granules and it so it could be high forward scatter high side scatter and then maybe i' have another cell that was Tin with lots of granules so it might be up here and I have another cell and I could actually go through and I would have a DOT for every one of my 100,000 cells telling me both how big and how granular that cell was scatter comp of cell just small guys two dimensional dot or Scatter Plots now let's see how laser light is used to detect individual cells In so here this is their side

scatter they've got the not so granular the medium granular the the very granular this is their forward uh they had forward scatter where it was kind of like mostly smallish a few really biggish and here they've put the same things on this on two uh graphs and so on two axes so they have really small stuff they've got some you know small stuff that's not super granular they've got some bigger stuff that's more Granular it's only a little bit bigger you know you can look at things in a lot of different ways like this and

each one of the dots on that plot is a single cell that we're able to get that on whoa this this video is going to make me mad again now let's see how laser light is used to DET individual cells in the forward scatter the onedimensional histograms we have seen so far do not necessarily show the complexity of the cell populations For example what appears to be a single population in the forward scatter histogram is reality multiple populations that can only be discerned by looking at the data in the second dimension this is done through

the use of two dimensional dot or Scatter Plots you can see that the Peaks from the forward and side scatter histograms correlate with the colored dots in the scatter plot now we can view the results obtained when we create a scatter plot Using forward and side scatter data from a typical peripheral blood cell run the populations that emerge include lympocytes which are small cells possessing low internal complexity monocytes which are mediumsized cells with slightly more internal complexity and neutrophils and other granulocytes which are large cells that have a lot of internal complexity this multi parametric

analysis is the real power of flow Cytometry now let's take a look at another parameter that no we're not looking at that parameter right now so this is actually some data that I generated um as a grad student um so this is blood um from um an animal where I just ran it and you can see all of the dots um i' have to look back at this uh to tell you how many exactly how many cells I ran that day we'll say it's a couple hundred thousand and so you can see all of them

you can also see that There are some places where there dots are kind of more denser than others that we often think about as populations of cells yeah there's like a weird cell that over here but there's also kind of places where there's big populations of cells and sometimes it's actually a little hard to look at you're like okay like is this area denser than this area or not I don't know at some point like it's just black um there are other ways you can actually make it look pretty Where you can have like Contour

plots of actually seeing how dense the dots are in different places or you can actually color code it based on density um where you can get some more more information about those plots um I know that there's uh definitely one paper that we're going to talk about this semester where they use Contours um I actually find Contours to be very old-fashioned um but it's old paper so um I like I use I like to use ones where you can actually see the Individual dots but you also get the colors to tell you how dense it

is and so if you look at this what you can notice is it looks like there's three groups in these cells yeah there are some weird outliers and I'm not going to try to figure out what those are unless like that was my experiment but they're just these three main groups um and I will um I can Actually even use my software and like draw a little gate around each of them draw a little region and the software will even say 60.7% of the cells were in this region you drew so 60.7 of the cells

are have this kind of phenotype 3.81% of the cells have this phenotype 28.4 have this phenotype and I can draw that so that starts to give me um pretty good information I could compare different Samples um this population of cells that is um sort of the smallest but totally the least granular those are lymphocytes so I know that this animal had 60. 7% of its blood uh it its white blood cells were lympocytes um 3.81% were monocytes because this is a known monoy population as sort of the largest ones that are sort of medium granular

this very granular kind of um mediumish size um are the granulocytes largely neutrophils because we're looking at blood here and So I can get that data in like a minute by just throwing my cells I didn't even have to put antibodies on but because I did put antibodies on them I can get some additional information because the laser light is going to interact with the dye that I put on the antibody and is going to excit it to emit light at a a known wavelength so I'm my laser light is basically going to Hit this

thing um the thing is officially called the fluorophor um and make it emit light and so I not only can I capture forward scatter and side scatter I can capture all the light that comes off of any of the fluorescence molecules that I have attached to my cell um this is the energy State diagram that they were going to tell you I guess I'll tell I guess how long Is it one of the most common ways to study cellular characteristics using flow cytometry involves the use florescent molecules such as Flor labed antibodies in these experiments

the labeled antibody is added to the cell sample the antibody then binds to a specific molecule on the cell surface or inside the cell finally when laser light of the right wavelength strikes the Flor a florescent signal is emed and detected By the flow cyer how is this fluoresent information collected the fluorescent light coming from labeled cells as they pass through the laser travels along the same path as the side scatter signal as the light travels along this path it is directed through a series of filters and mirrors so that particular wavelength ranges are delivered

to the appropriate detectors fluoresence data is collected in generally the same way as forward and S scatter data in a population of Labeled cells some will be brighter than others the voltage pulses are recorded and can be presented graphically and so in this case two-color experiment this case these cells only have a little bit of my protein of interest and these cells have a lot of my protein of interest um based on just sort of the histogram of amount of fluoresence of my protein of Interest how bright was it How much did we excite that

color and so we might well well for example talk about we're going to put an antibody with a green thing on it these cells only had a little bit of green the the antibody only bound them so that Target protein was only on them a little bit or was not on them these cells are very green so they have my target protein of Interest what if we want to do a two-color experiment if we analyze data from the Two-color experiment using a scatter plot four distinct populations emerge looking at the Dot Plot In terms of

quadrants cells with only bright orange fluoresence appear in the upper left quadrant okay so you saw this example where we're going to have these different colored things the example you just saw was green and was orange but it could actually be whatever colors my machine can read and so you when you buy your Machine no um when you buy your machine you might say Okay I want a machine that can read green uh yellow this orangey color and this pinky color yes the dyes have names which are labeled here you don't care about the names

of the dyes um and so then I could actually look at is my cell green and orange is my cell green and yellow is my sell green only is my sell none of the four and I can start to get all of this whether it's green or Orange or whatever it kind of depends on the machine I have and so if I used a machine that was sort of like this I would get for every cell for every one of my 100,000 cells I would get six measurements off that cell I would get a forward

scatter measurement I would get a side scatter measurement I would get a how much green I would get a how much yellow I would get a how much orange and I would get a how much red and so I could get all of That data on all of my cells within a couple minutes um and so hopefully you can see that that's super powerful um the cytometer that I have does not do four colors it's slightly less in four colors um the most complicated cytometers that I've ever used was a 19 color um there are

I I have generally said that there that the most complicated one that exists is 50 um but that's honestly not true and I know there are some even more crazy Complicated ones but I'm not going to deal because they start having other things added onto them and all sorts of bells and whistles so um like this four-color business is pretty often what we see and so basically we're getting forward scatter we're getting side scatter and we're getting whatever colors for each cell and then that's going into our computer to give us data on all of

those cells um what I also want you to notice Um is that after the cells go through this analysis they end up in the trash can so you can see my very well- drawn trash can here at the bottom um I will come back to that uh in a second and so um we can look at the data as you've seen in a couple different ways we can look at it with individual dots where we are actually picking any two parameters so here I basically picked orange and green and looked at myself and how much

orange and green They had and I said oh my gosh I've got four different groups based on high or low amounts of orange and green I can also just take them and be like okay I just want to know orange are they high or low on Orange and I can look at a cell count histogram or how high or low on day on green and do a cell count histogram so they showed you in the video how this can sort of like go side they they showed you histograms sort of going sideways and matching up

with a Dot Plot and I can't do that but if you need to see it again this these slides are posted and you can watch the video um and the other really cool thing that you can do is as I showed you you can actually draw little regions with your software and this software will tell you things like oh this percent of the cells are in the region that you just drew so you you said you care about cells with a forward scatter of 200 to 600 and a a Size scatter of less than this

you care about cells in this circle oh that's 60 60.7% of all the cells that that were in the blood so that's cool some interesting info but then you can do something else um officially these little regions or these little areas or groups of cells that I've drawn are called Gates I could if I looked at this say oh cool so I know those are lympocytes I know those are monocytes I know those Are granular sites I know that because I've done this a billion times um I actually today only care about lymphocytes I don't

care about the other ones I only care about lympocytes you know what I would do in that case I would double click on those lymphocytes and I could then take just the cells that were there and look at them on two other parameters of the many parameters I was looking at and I could say well of just the lymphocytes now That I've gated on the lympocytes so I am looking at only the lympocytes I can further subdivide the cells that were in this group and so this is actually a situation where I did that so

here I did further analysis of the lympocytes here I did further analysis of the monocytes here I did further analysis of the granula sites um looking for uh the expression of two other proteins on them um and you can see that by and large the Granulocytes didn't have those other proteins they were kind of low for this one and low for that one um but there were definitely four nice robust populations in my lympocytes and I could then say oh my gosh I only really like these lympocytes let me double click on that and examine

them with further stuff if I have a machine that does 16 colors I can like super finely distinguish all of these things and I could distinguish I look at these guys and then these guys And then these guys I could do like days of analysis of looking at different types of cells um and so you can see this here as well um so this might be you might say oh I really like this population of cells here this 24% I'm going to get rid of the other ones and only Analyze That 24% to see how

much of these two proteins cd8 and CD3 they have on their surface I only really care about the ones that have both so now I'm going to look at them further or H I Only care about these ones that have CD4 and CD3 so I'm going to look at them further um and so we can use our machine to actually subdivide that 100,000 cells and start to find out about amounts of different cells you can see that each time I'm getting a percent um which is basically what percent of the cells that that were in

this whole plot are in this gate that I've drawn so I can actually end up getting a ton Of information about this um this is what some actual photometry data looks like um and this is the uh another view of the same thing these are both very classic um this is a classic plot that Ian all a lot of IM all just know well um in when I was a second year grad student we always had this event in the second years had to design a t-shirt for everyone in the Immunology program and Every year

the second years would design a T-shirt and the person who was in charge of the program would throw it in the trash and design it himself because he hated whatever we drew so my class was like we're not putting time into this and so we we that's what we gave him is we gave we just gave him this picture and he thought it was the coolest thing he'd ever seen in his entire life um and so we all have pictures of so I now have a t-shirt with This randomly on it um it's it's something

that you see when you look at the thymus and so our class called it the thymus bird and said it was our mascot um so that's the thymus bird um and I also want to show you so I couple things that you can see here um often times when we look at plots like this though it doesn't have to be we tend to draw our gates like this where we make quadrants so we often will do it like this though it doesn't actually Have to be um we can see we get percentages of how many

cells are in each quadr let's imagine all right so this one we'll say is 80% this one's 12% this one's 3% let's imagine this one wasn't labeled because sometimes in some papers they don't label all of them usually the one they don't labels that one but let's imagine they didn't label this one is There a way we could figure out what percent of the cells were in this quadrant from the other information that we have I see lots of head shakes how would we figure that out yeah BR yeah if we have quadrants then we

know all they should all add up to 100 so we could just take a 100 and subtract each of these and find out how much is left if I wanted to know well how many Of these cells are have any cd8s on them at all all I might add up 80% plus 5% and say it's 85% um if I wasn't out of a 100 then I'd have to take 80 plus 5 divided by like all of them if I wanted to say how many of the cells that have cd8 also have CD4 I could do

80 divided by 85 um so I can get a lot of information from something like this um the and so you can see that basically when we do a Quadrant gate the bottom gate or the the bottom left a lot of times this semester we're going to see quadrant Gates um the bottom left is minus minus it's minus for both things sometimes we we might say low instead of minus but low or minus either way is fine so you can see that so in this case 3% were minus minus for the two proteins here we

have Plus for one of them minus for the other one plus Plus for cd8 which we can see from the axis Minus for CD4 it's about 5% 12% that are CD4 positive cd8 negative and 80% that are double positive for CD4 and cd8 so I'm again I don't care that you like know those numbers I care that you like know how to read them off of this graph if I were going to draw this on the board you guys are well aware of my awesome art skills and so I also want to show you how

I would draw this exact same plot um and one of the reasons I note this is because I'm going to be Drawing them a lot I also will ask you from time to time to draw them and so I want you to know the things that are sort of key in a drawing of one of these plots so if I draw that exact same plot I need to draw axes and label the axes so I get some partial credit just for that um and I'm not going to necessarily tell you you should have the quadrants

or you shouldn't I'll make them right now I Usually don't care if you actually have the quadrant lines or not but what you can see in this plot is that this population of cells the population of cells in this quadrant is pretty rare it's the smallest percent at 3% so I'm going to draw that as a small circle this one's 5% it's a bit bigger so I'm going to draw that as a Circle that's a little bit bigger this one's 12 so that's even a little bit bigger and this one's 80 so it's going to

be really big and so I'm usually going to be caring a lot that you have axes um that are labeled and that you have circles that and have appropriate sizes I'm not going to like have my millimeter ruler out and care about them if I was looking at this I would really care that this one was the tiniest and this one was really big and these were sort of like inish but I'm not going to like really get too deeply into it so That's what all I would want for a drawing of that plot sometimes

if a type of cell is not present in a particular situation there should be nothing in that quadrant so sometimes a quadrant could be blank that's okay um as I mentioned I have I have like one more thing I want to tell you about fetometry and then I'm just going to give you the problems um and you can work through all of the part one Problems you know what you need to know I'm happy to help you with them as much as you want the part two is going to be based on the data that

you generate next week when you do sociometry um so just so you know um this is actually what I did as a grad student is I took blood samples from mice every day after different infections and then I first got the lymphocytes and then looked at how many of the Lymphocytes had cd8 and how many of them could bind to my um antigen of Interest they had a specific kind of receptor on them and so I would just run cytometry on the blood every day get a percent and then put that on my graph and

I can get all sorts of fun graphs really easily this way um the f so there's one more slide and the one more slide is interesting to me in two ways one it is a modification of flow cytometry that Many people use and in fact people are using it more and more and more and more and even like not immunologists are using it all the time now so it's cool um it's really useful it it's also fun because it gets allows me to tell you a fun fact about all of this and I really enjoy

this fun fact um so some so I am always going to talk about all of this as flow cytometry some people talk about everything that you've seen here as Facts shown here at was said as facts if you hear about facts from people who are not being super picky like me they might mean flowy what I've told you guys as flow cytometry if we're being super technical fax is a specialized kind of flow cytometry and it's a specialized kind of flow cytometry called fluorescence activated cell sorting um so the idea with fluorescence activated cell sorting

is that you do All the things that we've been talking about with flow cytometry but there's an additional thing that happens at the end remember before the sample went into the trash can now the sample's not going into the trash can now here's our droplets with one cell per droplet that are coming out of the machine and when they come out of the machine they're actually going to get a electrical charge added to them so They're going to be positive or minus and the computer is going to figure out some information about them and I

am going to tell the computer I want cells that have these proteins on their surface I would like cells that have just protein a and protein B on its surface or I would like cells that have other things the computer is going to figure out what that cell is while the droplet is falling and then it's going to take These two plates here and switch around the electrical charges to make the droplet fall in a different direction and so in fact the cells can be sorted based on the proteins that they have um and I

can get collections of like cells with only one color or cells with two colors or cells with zero colors from sorting with my machine um this addition onto a machine is uh a makes it way crazy expensive um but you can get sorting and then you can take these Cells and go do other experiments with them um and so there are lots of situations where people will sort out particular types of cells from a homogeneous mixt or from a heterogeneous mixture um using fluoresence activated cell sorting do you have a question issues that comes with

that what kind of issues like are there things that to be able to use um me so yeah you do so you usually Do do it under some special aseptic conditions to deal with contamination and there are you do have to think a lot about your experiment and what you're doing afterwards and how you want to set it up so there there is a lot of planning that goes into all of that is what I will say on that so um but I still haven't told you the well I'm G to try one thing it's

probably going to fail and then after that I'm going to tell you my fun fact which is that once Upon a time somebody made a really cool video but I think if I remember correctly this link is dead but we're going to just try it just in case the link is done okay yeah um that's fine you can imagine that you don't need to see the video but I can still tell you my fun fact um so this these machines were being were invented in the late 7s early ' 8s um and at the same

time that this Machine was being invented there were some other machines that were being invented for not immunologists there's this one other machine that was invented for not immunologists for people who worked in random offices where they wanted were like Brena could have a piece of paper that she would like to send to someone in California through the phone so she could take her piece of Paper and she could put it in the machine and she could have it get sent to the someone else like in California through the phone and it would be a

copy of that piece of paper so what's another word for copy some of you might see where I'm going so yeah so how about I'm I'm going there okay yeah we're getting there you guys are right on the right track so it's because The word is honestly so oldfashioned you probably haven't thought of it so there's a word for copy that exists how the effect simile yeah so it's it's a it's a word that means copy and so they these people they made a machine so they could send people copies of the paper through a

pie through the phone line and they they were so excited they could send people a effect Sim they were so excited and let's imagine you invented a machine called a fact simile machine and that is actually the technical name of the machine is a faimily machine what would you call that machine that when you try to get a patent for it a fax how would you spell fax FACS right because that's the beginning effect simile guess what when they tried to get that patent the patent was already for FACS was already taken Because this was

invented the year before and so this took the patent for FACS fact and so the company who was doing all of this was Xerox and they were like crap we can't use FACS what are we going to use instead well there's a bunch of x's in our company's name so let's name it fact with an X instead because the our name that we want to use got taken so that's where the fax machine came from and why it's called facts and Why has an X in it instead of having a CS cuz we stole it

cuz we're awesomer I know you guys are like yes she is the nerd who was on Jeopardy um so those are my random facts about that um and I'm just going to give you guys this worksheet if you want to work on it now you should if you don't you don't have to but I'm happy to help you if you're working on it now um it's due in two weeks