Flowing in flow cytometry

On-demand webinar

Webinar objectives:

Part 1
- Identify forward and side scatter, gate strategy and compensation as key elements of flow cytometric analysis.
- Associate the importance of the different systems composing a flow cytometer and their different functions.
- Be able to apply the principles of flow cytometry to design an experiment.
Part 2
- Demonstrate a deeper understanding of all aspects of experimental design - materials, controls, reagents, color compensation, and multi-color design- to be able to develop complex flow cytometry experiments.
- Apply these advanced tips and tricks to get the best from your experimental design.
- Be confident in performing data analysis for your experiment.

Flowing in flow cytometry QA - Part 1

Video transcript

00:00 - 00:16: Thank you all for joining our live Q&A conversation about flow cytometry.
00:16 - 00:22: I hope you’re all having the chance to watch our two-part webinar series.
00:22 - 00:28: Feel free to submit any questions throughout the Zoom chat box throughout this conversation.
00:28 - 00:31: At this time, I’m joined by Dr. Sara Passos.
00:31 - 00:34: Sara, would you like to give a brief introduction of yourself?
00:34 - 00:36: Yeah, of course.
00:36 - 00:38: Thanks, Noah.
00:38 - 00:40: So guys, I am Sara.
00:40 - 00:46: I’m working at Abcam, a new development project department right now.
00:46 - 00:53: I worked for flow for the last eight years that I had been working with all different types of cells from animals, from human samples.
01:00 - 01:04: From animals, I have experience with rats, mice, monkeys.
01:04 - 01:11: With human samples, I have been working with some kind of infectious disease and healthy donors as well.
01:14 - 01:22: And during all these years, flow cytometry has become my life, as my professional life.
01:22 - 01:27: And I will be very glad to answer your questions.
01:27 - 01:30: And we will have, I think, 45 minutes, Noah?
01:30 - 01:31: Yeah, about.
01:31 - 01:32: Pretty much.
01:32 - 01:33: About this.
01:33 - 01:34: Okay.
01:34 - 01:37: Thank you so much, guys, for joining this meeting.
01:37 - 01:39: Okay, great.
01:39 - 01:42: So we’re going to start off with some questions that have already been submitted.
01:42 - 01:48: But again, feel free to submit any of your questions into the Zoom chat box.
01:48 - 01:55: Okay, to start off, flow cytometry is a measurement of what two things?
01:55 - 02:02: So flow cytometry, we can measure like with the light scatter by cells and the fluorescence from the fluorochromes that attach to the cells.
02:06 - 02:12: So that’s the reason that we have to use some fluorophores attached to the cells to bring the light into the instrument.
02:14 - 02:20: And you can read the cells using fluorophores.
02:20 - 02:23: Okay.
02:23 - 02:28: In flow cytometry, are you measuring all blood cells?
02:28 - 02:30: No.
02:30 - 02:31: Why not?
02:31 - 02:37: So we can use different types of tissues from, as I told you guys, from mice or from human samples or cultured cells as well.
02:41 - 02:48: So we can use a lot of different types, but we are not able to use all the cells.
02:48 - 02:49: Why?
02:49 - 02:51: Red blood cells, we have to lyse them.
02:51 - 02:56: So that’s the reason we are not able to use all the cells.
02:56 - 03:01: But we can use a lot of different tissues.
03:01 - 03:04: And why do you lyse the red blood cells?
03:04 - 03:10: So the thing is, we are not able to read the red blood cells if we are not lysing them.
03:10 - 03:17: For example, neutrophils, once you have whole blood and you have to purify the whole blood and separate these different subsets of cells.
03:20 - 03:26: So we have the PBMCs and we have the part of the blood that is on the bottom of the tube.
03:27 - 03:32: To do that, you have to lyse those cells with water, and then you’re going to have your neutrophils at the end of the process.
03:35 - 03:38: But you are not able to use them if you are not lysing them.
03:38 - 03:41: And once you lyse them, you break the membrane of the cell.
03:41 - 03:46: So you are not able to have anything attached.
03:46 - 03:47: Okay.
03:47 - 03:48: Okay?
03:48 - 03:49: Yep.
03:49 - 03:54: What is flow cytometry commonly used for?
03:54 - 04:01: So we use antibodies with fluorochromes to attach to the cells in order to recognize antigens in the cells.
04:04 - 04:10: And those antigens we can have, like for example, we have surface markers that the fluorophore recognizes the surface of the cells.
04:13 - 04:16: So we have all kinds of different stuff.
04:16 - 04:22: But the thing, the main thing for flow cytometry is to recognize antigens that are attached to those antibodies there.
04:25 - 04:26: Great.
04:26 - 04:31: It looks like we have a couple of questions coming in through the chat window.
04:31 - 04:33: Okay.
04:33 - 04:39: What factors have to be considered for detecting membrane proteins of low expression?
04:39 - 04:46: So you have to think about it that some fluorophores are brighter than others.
04:46 - 04:53: For example, PE, it’s one fluorophore that it’s the fetal aerogen.
04:53 - 04:54: It’s one that it’s very bright.
04:54 - 05:00: So if you are looking for something that is rare at the surface of the cell, it’s better to use some type of fluorophore that is very bright.
05:03 - 05:07: So it becomes easier to find with the instrument.
05:07 - 05:08: Okay.
05:08 - 05:09: That’s the main point.
05:09 - 05:10: Yeah.
05:10 - 05:12: That makes sense.
05:12 - 05:18: Did you also, what would your suggestions be for intracellular protein detection?
05:18 - 05:25: So intracellular is always a thing that people get a bit weird about it and just like scary because you have to rub the membrane and get the fluorophore inside.
05:33 - 05:37: And for this process, you have to fix the cell.
05:37 - 05:46: And this will make the process that the cells sometimes will not survive through the process because we use different reagents for that.
05:50 - 05:53: So it’s just like this.
05:53 - 06:03: So my main concern about using intracellular staining is about staining for a long time first.
06:04 - 06:10: So if you are able to stain just for a short time, like an hour, it’s going to be perfect.
06:10 - 06:16: I have already had the chance to use fixation for long periods of time without any trouble for my experiment, but it is a big concern about this.
06:21 - 06:25: So I never tell people to do that.
06:26 - 06:27: It’s better to use a short time.
06:27 - 06:29: Okay.
06:29 - 06:30: That makes sense.
06:30 - 06:31: So I think it’s better.
06:31 - 06:32: Right.
06:32 - 06:35: Okay.
06:35 - 06:36: So we have a couple more coming in through the chat.
06:36 - 06:37: This is from Zineb.
06:37 - 06:38: Okay.
06:38 - 06:41: I know there are different types of flow cytometry.
06:41 - 06:47: What are the purposes of the various fluorochromes, and how do you choose between them based on, how do you base that choice?
06:49 - 06:50: Yeah.
06:50 - 06:53: So we have to think about it.
06:53 - 06:59: As I mentioned during the webinar, we have different equipment, different instruments, and those instruments have different, for example, different lasers, different numbers of lasers.
07:04 - 07:05: So this will reduce your options of different fluorophores to use.
07:11 - 07:12: Okay.
07:12 - 07:14: So this is the main thing.
07:14 - 07:19: Once I mentioned to you guys that it’s important for us to know about the equipment that we have, it’s because of this, because then we’re going to be able to identify what options we will have in the end to do our analysis.
07:28 - 07:36: Sometimes we just make our panel of antibodies and fluorophores to use, but in the end, your equipment is not going to be able to read a specific channel.
07:40 - 07:44: So then you have to change your whole plan.
07:44 - 07:50: So that’s the reason I just say, first, take a look at your equipment, make notes, make sure that you have all the channels open that you needed, and then go ahead with your experiment.
07:56 - 08:03: And we have another question coming in.
08:03 - 08:08: Why do you need both an isotype and a negative control?
08:08 - 08:10: So this is the thing.
08:10 - 08:15: The isotype, it’s going to give you the negative control.
08:15 - 08:27: And as we already understand, what I do during my experiments is I use isotype control as the negative control.
08:27 - 08:33: And I still have some negative cells for my experiment.
08:33 - 08:41: It’s not a control inside of the experiment that I’m doing.
08:41 - 08:47: So I have the option to use, for example, positive and negative cell lines for the same type of staining.
08:48 - 08:54: And I still use the isotype controls as my negative control for the experiment.
08:59 - 09:00: So it’s the negative control inside of the experiment for the whole thing, for the negative and the positive.
09:00 - 09:04: I don’t know if that was completely clear.
09:04 - 09:05: No, that makes sense.
09:05 - 09:11: So if something goes wrong with the isotype control, what would that mean for the experiment?
09:11 - 09:17: So if something goes wrong with the isotype control, it means that you have to make sure about the positive staining that you were using.
09:22 - 09:28: Because the isotype control is going to identify where the negative for that specific fluorophore is.
09:29 - 09:35: And if you don’t have there, it becomes difficult to identify where is the positivity and where is the negative shift.
09:37 - 09:39: Okay, great.
09:39 - 09:41: That makes sense.
09:41 - 09:42: Yeah.
09:42 - 09:43: Okay.
09:43 - 09:47: This is a question coming in from Carlos.
09:47 - 09:54: Are there two types of cytometers: by three differential and five?
09:54 - 09:58: Do they use different types of fluorochromes?
09:58 - 10:00: So they can.
10:00 - 10:03: They can use different types of fluorophores.
10:03 - 10:09: For example, sometimes you have one channel that is open and you can use all types of APC.
10:11 - 10:14: But some instruments don’t have the UV laser.
10:18 - 10:22: So you’re not able to use the BUVs.
10:27 - 10:29: So that’s the reason that I’m telling you that you need to know the characteristics of the instrument before trying to plan any experiment.
10:29 - 10:33: Okay, great.
10:33 - 10:35: Yeah, that makes sense.
10:35 - 10:36: Yeah.
10:36 - 10:41: Could you explain what happens inside the flow cytometer?
10:41 - 10:46: During the experiment or because it’s like once you have…
10:46 - 10:54: So it doesn’t matter if you have tubes or plates, whatever you are reading your samples.
10:54 - 10:58: So you’re going to have the laser, it’s going to hit the cell.
10:58 - 11:03: So it’s the first thing that occurs inside of the equipment.
11:03 - 11:09: So we have the laser that hits the cell and this has to be one-by-one cell passing through the laser.
11:11 - 11:16: And that’s important because if you have two cells attached, the first parameter that is measured inside of the flow cytometer is the FSC and SSC.
11:23 - 11:27: This means the size of the cell and the granularity of the cell.
11:27 - 11:32: If you have two cells passing through the laser, you’re going to have double the size, for example.
11:33 - 11:34: But this is not true.
11:34 - 11:39: So it’s the first thing that happens inside of the cytometer.
11:39 - 11:44: After that, as the cells pass through and collect this information first, you will have a complex thing that is the second part of the cytometer.
11:53 - 11:56: Because we have three parts, as I mentioned in the webinar.
11:56 - 12:00: So we have the optics, the electronics that are very, very important.
12:07 - 12:08: And we have a collection of filters, mirrors, and that is the concern of all these cytometer things.
12:08 - 12:13: That is, let’s say, the brain of the instrument.
12:17 - 12:23: And one thing just passes after the other.
12:23 - 12:27: Once the one color hits the mirror and goes through the filter, then you have one color read and one information collected.
12:27 - 12:33: Then if it’s higher than this, it goes to the other mirror and then to the other filter again.
12:34 - 12:38: And it’s going to collect another color after that.
12:42 - 12:46: Because of the wavelengths being different, and it collects one after the other until the end that all the data is collected into the computer.
12:46 - 12:47: That is the electronic part.
12:47 - 12:48: Okay, great.
12:48 - 12:49: Yeah, that makes sense.
12:49 - 12:55: I don’t know if it was clear enough, this answer.
13:02 - 13:04: But if you guys want some more clarification, I can take notes and send by email later on.
13:04 - 13:08: Yeah, that was great.
13:08 - 13:10: Okay, we have another question coming in from the chat.
13:10 - 13:17: It’s a little bit specific.
13:17 - 13:24: If isolating immune cells from a mouse primary liver, say from a disease and a control animal, if the viability of the cells is 85% in the control liver and the viability is 15% in the disease model, will this matter for your downstream multicolor flow if the starting numbers and ratios are not the same?
13:30 - 13:36: Yeah, so that’s the big thing.
13:36 - 13:42: You have to pay attention to the number of events that you will collect during your experiment.
13:43 - 13:48: Usually, we collect 10,000 events here.
13:48 – 14:01: But if you are looking for something that is more rare, or you don’t have enough cells or we don’t have enough markers for that, you are looking for something one thing or the other.
14:01 - 14:07: So my tip is just to try to get as much as you can.
14:08 - 14:18: And in the end, probably you have to do some calculations to just have the same base for all of them, the sick one and the healthy one, the control.
14:19 - 14:30: Because you are not going to be able to compare and say, okay, we just have two cells stained in the sick mouse, but you have 10,000 in the control.
14:30 - 14:35: But if you started with a high number in the control, of course, this comparison will not be able to be done like this.
14:43 - 14:44: Great.
14:44 - 14:45: Okay.
14:45 - 14:46: Great.
14:46 - 14:47: Yeah.
14:47 - 14:52: So can you briefly describe compensation and dealing with compensation when you’re working with one or more wavelengths?
14:52 - 14:54: Yes.
14:55 - 15:00: So compensation is a big trick for everybody.
15:00 - 15:07: Everybody is scared about compensation all the time once people start talking about flow.
15:07 - 15:09: And compensation is just math.
15:09 - 15:11: It’s nothing but this.
15:11 - 15:20: So we just have to make sure that once we have our fluorochromes already planned to use, this is going to give you a lot of information because the wavelengths are different.
15:25 - 15:32: And once we have, for example, the FITC color and the PE color, for example, and you run your sample, these are completely different wavelengths.
15:35 - 15:38: They are not overlapping one to another.
15:38 - 15:45: This gives you a clearer way to have your compensation well done because they are very separated one from another.
15:45 - 15:47: So once you have this, it’s clear to identify where your population is in different colors and different, of course, wavelengths.
15:56 - 16:01: So compensation is pretty much one color minus the other once they overlap each other.
16:01 - 16:02: Once they are not overlapping, it’s very separated subpopulations of cells.
16:07 - 16:11: So it becomes clearer to do it like this.
16:11 - 16:18: But of course, the majority of the time, we don’t have the option to have just two or three colors.
16:18 - 16:20: So we have to use more.
16:20 - 16:27: I had experience that I did an experiment with some mouse samples that I had to use 16 colors.
16:27 - 16:34: And of course, in this case, we have a lot of overlaps, and we have to compensate all of them.
16:35 - 16:38: And it’s just one minus the other.
16:38 - 16:41: And you’re going to have, in the end, all the clarifications of the subsets of cells there.
16:42 - 16:49: And is that the same as running the FMOs, or is that a different process?
16:49 - 16:50: Exactly.
16:50 - 16:54: So compensation, you can use different ways to do it.
16:54 - 17:02: You have to use single color staining that you use one tiny amount of sample with each color.
17:03 - 17:10: And once you have a lot of multicolor, as we call, experiments, you have to use FMOs.
17:10 - 17:12: That is fluorescence minus one.
17:12 - 17:17: So you can put all the fluorophores in one tube minus one.
17:17 - 17:25: So this gives you the opportunity to identify all the colors minus one in each tube.
17:25 - 17:29: So you can use FMOs, you can use single colors.
17:29 - 17:31: You have to use your isotype control.
17:31 - 17:35: That’s the important thing because you’re going to identify where the negative part of the fluorophore that you don’t want to be inside that.
17:35 - 17:40: So with all these together, you are able to compensate your experiment.
17:45 - 17:50: This means telling the equipment where each color has to be because the equipment doesn’t know.
17:51 - 17:55: And you have to give this information that it’s numbers.
17:55 - 18:00: And the numbers will give you the color in the end, let’s say like this.
18:00 - 18:01: Okay, great.
18:01 - 18:02: Yeah, that makes sense.
18:02 - 18:06: So we have a couple more questions coming in.
18:06 - 18:08: This one’s from Kelly.
18:08 - 18:13: When you’re working with different antibody reagents for your specific cell type you want to identify, could you go over a couple of circumstances that might make understanding results difficult?
18:20 - 18:22: Yeah.
18:22 - 18:25: So Kelly, here is the thing.
18:25 - 18:26: Once we are using…
18:26 - 18:30: So we have different protocols, different procedures depending on the tissue or the type of cells that you are using.
18:33 - 18:34: Why is this?
18:34 - 18:40: Because we have different tissues that are hard to disaggregate the cells.
18:40 - 18:46: And to do this, we have to use different reagents.
18:46 - 18:54: So once we have, for example, I had experience doing an experiment with arteries.
18:58 - 19:01: So we have cells that are living inside of the arteries.
19:01 - 19:07: And those are very difficult to find out.
19:07 - 19:12: So because of this, they are such a tiny amount, and we don’t want to lose them.
19:12 - 19:18: So for this reason, we have to use reagents that are, let’s say, soft.
19:18 - 19:24: They are not killing the cells; they are not making any changes at the surface of the cells because we know that different reagents, for example, for fixation, they can change the cell membrane, and we don’t want this.
19:30 - 19:36: So it’s very important to know what type of tissue, what type of cells are you going to use.
19:36 - 19:37: And this will give the information about the type of reagents that you should use as well.
19:43 - 19:50: So this is going to give you an idea. Once you are using, for example, cells from PBMCs, so they are more resistant to the reagents.
19:54 - 20:01: So we can use things like PBS or we can use FACS buffer, and they still survive no matter what.
20:05 - 20:06: You can fix them.
20:06 - 20:12: I have already had experience fixing them for more than 20 days, and they still are perfect.
20:13 - 20:19: So depending on the type of the cell, you have to take a look at this, read about it, try to reach other people, other researchers, or people that will be doing the same type of experiment like you and try to get this information out.
20:31 - 20:32: Great.
20:32 - 20:33: Okay.
20:33 - 20:41: Here’s another question from Chiara, and this is stepping back to the FMO.
20:41 - 20:45: If you use the FMO, do you need the isotype control as well?
20:45 - 20:47: Do they not tell the same information?
20:47 - 20:48: Okay.
20:48 - 20:58: So Chiara, I always run everything that I can, including isotype control, FMOs, single color staining, unstained samples, because these will give me the opportunity to play around with the results that I will get in the end.
21:12 - 21:17: The majority of the time, if you are using something that is rare, you are looking for something that is rare inside of your cell or surface of your cell, or if you are looking for a specific type of cells during infection, for example.
21:28 - 21:35: So if you have all these options done, it’s not like a thing that you have to do, but if you can do it, it’s going to be better for you because it’s going to clarify a lot of questions that may come out in the end of your experiment, and you are not going to be able to go back anymore.
21:45 - 21:48: So if you can do it, it’s better.
21:52 - 21:53: That’s my advice all the time to people.
21:53 - 21:55: And it was the way that I learned.
21:55 - 21:59: So I prefer.
21:59 - 22:05: And when you run it, will you do all the controls every run if you’re running the same panel over and over again, or is that more for just setting up the panel in the beginning?
22:06 - 22:07: It depends.
22:07 - 22:36: That’s the thing, Noah, because if you are running in one day your whole panel, and if you need to run again, but the following week, you have differences in the equipment, like you have to learn your CST beads in a way to get your equipment regulated, like the lasers, the intensity of the lasers and everything.
22:36 - 22:46: So if you run in one week, and the week after, nobody is 100% sure that it’s going to be the same parameters, the same numbers.
22:46 - 23:00: So if I run the same antibody, the same lot, everything the same, the same type of cell, like a lineage of cells that are purified from whatever, and you’re going to use the same, the parameters will be different.
23:01 - 23:07: This, it’s like, it’s going to be at least like one parameter will be different.
23:13 - 23:19: So I all the time do everything from scratch.
23:19 - 23:23: But if you can do it with the same panel, that’s the thing that the equipment right now do the automatic compensation.
23:30 - 23:34: So you can use, of course, the same panel that you used two days ago; you can use the same panel two days after.
23:39 - 23:40: But if something goes differently, you don’t know if it’s because of the equipment or because of your sample.
23:41 - 23:42: Great.
23:42 - 23:43: Yeah.
23:43 - 23:44: That makes sense.
23:44 - 23:55: Do you have any recommendations of good resources for beginners who are just getting started?
23:55 - 24:00: First time using flow cytometry or just designing their flow panel?
24:01 - 24:08: So that’s the thing, guys, flow, you just learn once you’re doing it every single day.
24:14 - 24:18: You can read it, you can have all the information, all the theory, it’s going to help, of course.
24:22 - 24:24: You have to learn about the instrument, as I told you before, at the beginning of this question and answer.
24:28 - 24:29: But it’s just like doing day by day.
24:30 - 24:39: And it’s like asserting and then having mistakes is going to be part of the game, you know, and you’re going to learn a lot from this.
24:40 - 24:44: So until nowadays, I’m learning tons of things that I never saw before.
24:45 - 24:46: Because it happens all the time.
24:47 - 25:00: So we have to, of course, we can read a lot of things, have a lot of materials around, and I can send through email, a lot of references to you guys.
25:00 - 25:02: But you have to do it.
25:03 - 25:08: And about the panel, the first thing is to know the instrument.
25:09 - 25:12: That’s the first question; you have to know your instrument.
25:13 - 25:19: And after this, you’re going to collect all the information, like all the options that you have.
25:19 - 25:25: And based on this, you go to your material, your cells, your tissue, what do you have to say?
25:25 - 25:29: And you’re going to put one beside to the other and see your options.
25:29 - 25:33: Then it’s your first step to plan your experiment.
25:34 - 25:39: Great, thank you. Here’s another question that just came in.
25:40 - 25:54: When trying to identify cell phenotypes, for example, macrophage phenotypes within a sample that have a lot of overlap of markers, do you have any recommendations for navigating this?
25:54 - 26:03: Yeah. So I worked with macrophages a couple of years ago, and I was working with macrophages with human samples.
26:04 - 26:10: Last year, two years ago, actually, I had this experiment with lung macrophages.
26:10 - 26:15: So depending on what type of tissue your macrophage is coming from.
26:15 - 26:22: So I don’t know if your macrophage is from blood, your macrophage is from liver or lungs, whatever.
26:23 – 26:30: So they have different markers and different ways to set up your experiment for that.
26:30 - 26:40: You can tell me later on what type of tissue you are using, or if it’s from blood, whatever, and I can try to help you with that.
26:40 - 26:51: Because for lungs, for example, I used 12 different colors, different fluorophores to identify macrophages from lungs because they are very specific.
26:52 - 26:57: But if it’s from blood, you can use different ones.
26:57 - 27:05: And it depends on what type, if it’s from a human or mouse or rats as well.
27:07 - 27:09: So macrophages are a little tricky.
27:12 - 27:14: Right.
27:14 - 27:17: So the more overlap, you just have to add more colors and gate more.
27:21 - 27:23: Yeah, exactly.
27:23 - 27:30: So in the end, you have to start gating with FSC and SSC, and then you go for your single cells, and then you go for CD14, CD16, and then you go for…
27:35 - 27:38: So it’s like a whole way to walk through until you find whatever you were looking for inside of the macrophage panel.
27:42 - 27:49: So yeah, it’s a lot of things that you have to look for depending on the source.
27:49 - 27:54: This person actually just got back again and said that macrophages are from either peripheral human monocytes or they are tissue resident from bone tissue.
27:55 - 27:56: Okay.
27:56 - 27:57: Yeah.
27:57 - 28:01: So it’s like, depending on where, if it’s from one source or the other.
28:01 - 28:05: So you can use different markers for each one.
28:05 - 28:18: So and of course, the colors, the fluorophores, the markers will just overlap one to another, but there are ways that you can identify and separate them.
28:18 - 28:36: You just have to do it a lot of times, and you’ll be able to, for example, for me nowadays, I can identify neutrophils by eye, but of course, I do stainings, and I have different stainings for them.
28:36 - 28:41: So once I have the staining, I know where they are for sure, because I have to prove to other people where they are.
28:42 - 28:46: It’s not my eye that is going to give this information to them.
28:47 - 28:48: Right.
28:48 - 28:49: Yeah.
28:49 - 28:51: Great.
28:51 - 28:52: Here’s another question that just came in.
28:53 - 29:00: When extracting cells from biomaterials through biomaterial digestion, will this impact the cell markers that will then make flow difficult?
29:07 - 29:22: So what I can say for that is it can impact, as I told you. Once during your procedure, your cells can suffer; the membrane is the part that is very delicate of the cell and can suffer with that.
29:22 - 29:34: But this will not affect the staining because whatever is on the surface, the antigen that is there, the antibody will attach to that.
29:34 - 29:37: So it’s going to be there.
29:37 - 29:41: You cannot kill the cell because of course, this is not going to give you the information that you want.
29:43 - 29:45: But apart from this, it’s fine.
29:48 - 29:48: Great.
29:48 - 29:48: Thanks.
29:50 - 29:52: Here’s a question that just came in from Tyler.
29:53 - 29:58: Once I run my experiment and I perform the analysis, the data looks very different from one graph compared to another.
29:58 - 30:00: Why does this happen?
30:03 - 30:04: So Tyler, that’s the thing.
30:05 - 30:11: We have different ways to look at our data, actually.
30:11 - 30:15: And of course, each one comes for a reason.
30:15 - 30:22: They are not creating different types of graphs just to be nice to us.
30:22 - 30:24: No, this has a reason.
30:24 - 30:29: So the way that it is, it’s like if you are looking for two different parameters only, you use the graph that is going to be able to identify those two.
30:34 - 30:40: If you are using something that is rare, should I use dot plots or should I use another thing, another way to look at?
30:42 - 30:50: If I’m going for some cells that it’s highly expressing something, so sometimes you may go for dot plots; it’s too much.
30:52 - 30:57: It’s like a group of plot dots there until they are plotted, so it doesn’t look good.
30:58 - 31:03: And that’s the reason that each one looks different because of this.
31:03 - 31:10: So they are made for a purpose that is not random.
31:10 - 31:10: It’s not just to look nice to our eyes.
31:10 - 31:17: So I think the last part of the second part of the webinar is exactly about this.
31:17 - 31:18: So it’s about data analysis.
31:18 – 31:27: So if you guys want more information, I can pass through but I think I put it there the majority of the things that it's involved on data analysis.
31:27- 31:29: Great.
31:29 - 31:29: Thank you.
31:30 - 31:30: Welcome.
31:31 - 31:36: Could you explain a little bit about what you need to understand about the engineering of these big machines?
31:36 - 31:37: Obviously, some of the flow cytometers can be a little intimidating when you first start working with them.
31:41 - 31:42: So what do you really need to understand about them?
31:45 - 31:46: Yeah.
31:46 - 31:54: So the major thing that you need to understand, it’s like the wavelength.
31:54 - 31:56: They are the most important part.
31:56 - 31:58: So we need to understand.
31:58 - 32:00: Why do we need to understand this?
32:00 - 32:05: Because this is going to give you a lot of confusion or a lot of qualifications.
32:05 - 32:11: Since the beginning about the staining, as I told you guys, so we need to know about which colors we are able to use.
32:14 - 32:19: And in the end, it’s like the difference of the wavelength that this equipment will give you.
32:19 - 32:25 : It’s going to give you the possibility to do the compensation as clear as you can.
32:25 - 32:29: Or you’re going to just mess it up with your experiment.
32:30 - 32:36: But of course, flow cytometry is a technique that has this equipment.
32:36 - 32:40: And nowadays we have on the market a lot of different ones.
32:41 - 32:45: And I think in the near future, it’s going to be an equipment that it’s going to run an experiment by itself.
32:45 - 32:47: You won’t even need to turn off the equipment anymore.
32:50 - 32:56: So you just leave it there running like 40 plates, and they will turn it off, and you don’t need to be there.
32:58 - 33:00: But of course, all the analysis, you have to do it.
33:01 - 33:07: And because of this, it’s important to understand how the equipment works.
33:09 - 33:13: Nowadays, of course, we have problems with equipment that one day doesn’t work.
33:14 - 33:15: It seems to be that those equipment doesn’t like Mondays.
33:17 - 33:21: So it’s like Monday morning, you will go there, turn on your equipment, and it doesn’t work.
33:25 - 33:27: And you are just freaking out because you have a huge experiment to run.
33:27 - 33:33: Sometimes it’s a tiny thing.
33:34 - 33:38: It’s just a matter of a clog in your equipment that you can fix without asking like an engineer or a tech to come to your site.
33:39 - 33:42: Some stuff like lasers, never touch them.
33:42 - 33:46: So, or you’re going to kill your equipment forever.
33:50 - 33:51: So, but apart from these little things, it’s just like washes.
33:51 - 33:57: Just wash your equipment and well-maintain it, and it’s going to be nice forever.
33:57 - 34:00: Great.
34:01 - 34:03: Here’s another question that came in from Kiara.
34:04 - 34:20: If you want to block Fc receptors in a nonspecific staining, would you incubate your cells with the Fc antibody before all the other antibody markers in your panel, or would you incubate it with the other antibodies and stain everything at the same time?
34:21 - 34:23: So Kiara, thanks for your question.
34:24 - 34:39: I do Fc before the other stainings that I need because if you block the Fc first, once you get your staining done, this is not part of your headaches anymore.
34:40 - 34:41: So do it first.
34:43 - 34:43: Great.
34:43 - 34:44: Yeah, that makes sense.
34:44 - 34:45: Yeah.
34:46 - 34:48: Here’s another question that just came in.
34:48 - 34:53: I have a few experiments or I have a few experiences with flow cytometry.
35:54 - 35:03: Just being beside my colleagues, what should I keep in mind when I’m trying to use primary cells specifically?
35:03 - 35:06: Are there differences compared to cell lines?
35:08 - 35:09: Yes, of course.
35:09 - 35:12: Cells are always different from one another.
35:13 - 35:22: And we have to keep in mind, for example, if you are using lineage of cells that you bought from a company and came to you frozen.
35:23 - 35:29: So these cells have to go through a process to thaw.
35:29 - 35:33: So this can give you some differences.
35:34 - 35:44: Comparable to cells that you have, for example, from fresh blood that you already got and you just purify and do it.
35:45 - 35:53: Because those cells can be dead or can be apoptotic right now compared to the others.
35:53 - 35:56: So that’s the difference.
35:56 - 36:00: But related to staining, related to surface markers, no.
36:02 - 36:06: So T cells is always T cells, even if they are fresh or not.
36:06 - 36:10: If they are from the same source, they are always the same.
36:12 - 36:17: If you are having controls from the same type of animal or human, whatever, they are always the same.
36:20 - 36:20: Great.
36:22 - 36:24: This one is from Sasha.
36:25 - 36:29: During your presentation, you talked about different reagents used to set up the experiment.
36:29 - 36:34: Could you clarify on this and maybe just talk about what those different reagents are?
36:35 - 36:36: Yeah, Sasha.
36:36 - 36:38: So that’s the thing.
36:38 - 36:40: You can use FACS buffer, for example.
36:41 - 36:51: You can find those online from different companies that sell it ready to use, or you can prepare your own.
36:51 - 36:57: And of course, depending on the cell, you’re going to use different reagents to make your FACS buffer.
36:57 - 37:03: That is going to be the one that you’re going to use to dilute your antibodies for your stain.
37:05 - 37:09: Or you can use, for example, PBS 1X.
37:10 - 37:16: And I know a lot of people that use PBS to wash the samples and to do the staining as well.
37:17 - 37:18: This is going to hurt.
37:19 - 37:19: Probably not.
37:20 - 37:25: But depending on the type of the cell that you are using will make a little difference.
37:26 - 37:33: So that’s the reason that we always have to read the data sheet about the reagent, about the antibody that you are using.
37:35 - 37:43: And then what the company, what the manufacturer tells you about it, what you should do.
37:43 - 37:49: And if this reagent that they are saying to you to dilute your antibody is going to hurt your cell in a way that you’re going to lose your sample.
37:54 - 37:58: The majority of the time, this has never happened in my hands, but can happen.
38:00 - 38:02: So it’s like you have to find another way.
38:03 - 38:19: And, but that’s the reason we are always reading about everything once you are planning your experiment about your reagents, about your antibodies that you’re going to use, your fluorophores, and everything before you start to do it.
38:20 - 38:23: Because once you did, you cannot go back.
38:24 - 38:28: Great.
38:28 - 38:29: Here’s another one just coming in.
38:30 - 38:33: Flow cytometry has some different applications.
38:34 - 38:40: Could you explain what the different applications are and how you should change your protocol depending on the applications?
38:41 - 38:41: Yes.
38:41 - 38:43: So that’s the thing.
38:43 - 38:51: If you are looking to characterize a cell that is in your hands, you’re going to use the phenotype.
38:52 - 38:54: You’re going to just phenotype those cells.
38:54 - 39:01: This is going to be a completely different protocol if you want to find an antigen that’s inside of the cell.
39:03 - 39:08: Because one protocol doesn’t need fixing and permeabilization.
39:09 - 39:13: The other one that you need to look inside of the cells will need to do this.
39:22 - 39:24: Sometimes you just have a sample that you can stain, but you were not able for some reason to run on that day.
39:28 - 39:30: Or your equipment was broken, it had some problems, had some clog that you were not able to fix.
39:35 - 39:37: So you have to fix instead of trashing your whole experiment.
39:39 - 39:41: So this, it’s another procedure.
39:43 - 39:43: And sometimes you just run nice and smooth.
39:43 - 39:49: So you have differences between what type of thing you are looking for.
39:49 - 39:52: If it’s something on the surface, if it’s something inside of the cell.
39:53 - 40:06: And of course, depending on the type of reagents that you were using for that, because different cells, different aims for your experiment, it’s going to give you different procedures, protocols for that.
40:08 - 40:09: Great.
40:09 - 40:10: Yeah, that makes sense.
40:12 - 40:14: Here’s a question coming in from Louisa.
40:15 - 40:19: For the compensation panel, what do you think is better, using beads or cells?
40:20 - 40:22: The sample that she’s working with is fresh blood.
40:23 - 40:24: Okay.
40:24 - 40:28: So Louisa, for compensation, beads look wonderful.
40:29 - 40:33: So if you can use beads, I will say use the beads.
40:34 - 40:41: But sometimes we have some staining that it’s not able to do with the beads.
40:42 - 40:43: What does this mean?
40:43 - 40:55: So we have antibodies that are mainly against like anti-rabbit, anti-human, anti-mouse, anti-rabbit.
40:55 - 41:05: But if you read, depending on the beads that you have, if you read the data sheet of the beads, they will say exactly what they are able to recognize.
41:05 - 41:11: And sometimes they are not able, for example, to be stained by anti-rabbit.
41:12 - 41:17: So then you have to use cells instead of beads.
41:23 - 41:24: But depending on what type of experiment you’re running for compensation.
41:26 - 41:27: Great.
41:27 - 41:28: Thanks.
41:30 - 41:30: Here’s a question from Naren, actually talking about working with rabbits.
41:30 - 41:31: Okay.
41:30 - 41:36: I’m working with rabbits now and finding it hard to get antibodies against rabbits as most of them are raised against rabbits.
41:38 - 41:39: Again, yeah.
41:39 - 41:43: Is it possible for you to go through a rabbit spleen cell?
41:44 - 41:47: Are there any differences compared to mice or humans?
41:49 - 41:50: So that’s the thing.
41:50 - 41:01: That’s an issue that a lot of researchers have nowadays because of this, like the majority of the antibodies are made in rabbits.
42:02 - 42:08: We have a huge amount that’s from mice as well, but rabbits, it’s the majority of them, I think, nowadays.
42:11 - 42:16: And of course, there are differences between the spleen from rabbits compared to humans or mice.
42:18 - 42:24: And you are not able to use, for example, the same type of reagents between them.
42:25 - 42:32: So you still have to look for something.
42:32 - 42:42: Sometimes, for example, once I was working with monkeys, I can use some antibodies that were anti-human because we are buddies.
42:42 - 42:45: We are very close to one another, so we can use.
42:45 - 42:53: But it’s not a thing, if someone will question you, then you have to make sure what you use for that.
42:54 - 42:55: So, yeah.
42:56 - 42:56: Right.
42:56 - 42:59: Just some extra work in setting up the panel.
43:00 - 43:01: Yes, of course.
43:02 - 43:02: Okay.
43:02 - 43:04: Here’s another question coming from Naren.
43:06 - 43:12: Can the spleen cells be stored in liquid nitrogen and after a few months, thawed, then used in flow?
43:12 - 43:13: If so, what is the procedure?
43:14 - 43:15: Yes.
43:17 - 43:26: So we can just freeze tissue cells, whatever, and we can thaw them to use.
43:26 - 43:38: Depending if you were buying or if you have it in-house, depending on the type of experiment, for example, I don’t know if your spleen is from an infected animal.
43:38 - 43:41: I don’t know if your spleen is from a healthy animal.
43:43 - 43:53: You can use the same procedure; you’re going to find online probably a lot of protocols about how to thaw spleens.
43:54 - 43:56: And it’s not difficult at all.
43:56 - 44:05: It’s just like you take it out from the liquid nitrogen, you have to wash it because you want to keep your cells more healthy after that.
44:06 - 44:21: So you just need to wash it and then keep them probably in media, whatever media you’re using for a couple of hours and make them healthy, like breathing and alive.
44:21 - 44:26: And then you can just try and do your experiment with the same protocol that you have.
44:27 - 44:30: But it’s not a specific thing at all.
44:30 - 44:40: But I have some of them, so I can send to you for this person in Iran if they want it.
44:40 - 44:41: I have some type of different ones.
44:42 - 44:42: Oh, great.
44:45 - 44:45: Okay.
44:45 - 44:46: Here’s another question from Iran.
44:47 - 44:48: Okay.
44:49 - 44:52: How do we know exactly the amount of gating needed?
44:52 - 44:55: Could you also just kind of explain gating a little bit?
44:56 - 45:01: And also, how do you know when you’ve done enough compensation?
45:03 - 45:04: That’s the thing.
45:04 - 45:11: The amount of gating needed doesn’t have a rule; it doesn’t have a number.
45:12 - 45:18: First, because, for example, NK cells, we just have between 5% and 10% of the whole PBMCs.
45:20 - 45:25: For the other side, T cells, we have 37% to 53%.
45:26 - 45:32: So we have a lot of differences between the amount.
45:32 - 45:38: So once you gate your cells, it’s going to be different, and it’s going to be harder.
45:38 - 45:44: So you have to make sure that you have the correct stain for each population of cells.
45:46 - 45:48: And then this is going to be good.
45:48 - 45:52: And draw the gate, it’s based on the markers that you have.
45:53 - 46:00: That’s the reason it’s like make as much stain as you can in a way to have the better gating strategy for that.
46:02 - 46:03: Okay, great.
46:04 - 46:15: You’ve talked about this a little bit in your video, but I’ve seen some different gating strategies with lines, making different quadrants, and then sometimes circling the different populations.
46:15 - 46:19: Could you just talk about the advantages and disadvantages of those?
46:19 - 46:20: Yes.
46:20 - 46:21: So that’s the thing.
46:21 - 46:33: If you draw like a gating strategy around your population, you’re going to have a more clear gating strategy for that one.
46:33 - 46:38: Because once you draw just the quadrant, I will do this in the next panel.
46:39 - 46:51: Because if you are looking, for example, T-cells, you draw your gate around exactly the T-cells, and the next panel will show up, for example, CD3 and CD4s.
46:51 – 47:00: So this, you can draw a line in your quadrant and it’s going to separate the differences between those subsets.
47:00 - 47:05: But first, I will draw the gate exactly in the population that you are looking for.
47:07 - 47:08: Okay, great.
47:08 - 47:11: Thank you, Sara, and thank you everyone for attending.
47:11 - 47:14: We are finishing up our time right now.
47:15 - 47:17: I’m very sorry if I didn’t get to everyone’s questions.
47:17 - 47:25: Feel free to submit any further questions you have to events at abcam.com, and we’ll get to those, and Sara will get back to you.
47:28 - 47:28: Yeah.
47:28 - 47:30: Thank you so much, Noah.
47:30 - 47:31: For being here with me today.
47:32 - 47:34: Thank you for all the questions, guys.
47:34 - 47:38: I hope I were able to answer everything.
47:39 - 47:46: If I was not able to answer exactly what you needed, you can, as Noah said, email, and we can clarify as much as I can.
47:50 - 48:02: But try to be safe and thank you so much.

Flowing in flow cytometry - Part 2

Video Transcript

00:00 - 00:11: Hello, welcome to Abcam’s webinar, Flow Cytometry, Part 2.
00:11 - 00:14: During this webinar, we will cover the following objectives.
00:14 - 00:20: Be able to design and plan an experiment for flow cytometry, run your own experiment at
00:20 - 00:27: a flow cytometer based on the design and plan, and perform a data analysis of your experiment.
00:27 - 00:30: Thank you and I hope you enjoy.
00:30 - 00:31: Thank you for joining me today.
00:31 - 00:41: I will talk about how to plan and design your own experiment in flow cytometry.
00:41 - 00:46: Based on the first part of this webinar, I think we already have the tools to go into
00:46 - 00:49: this second part.
00:49 - 00:56: So I plan to start my presentation today at the second part of the webinar, talking about
00:56 - 00:58: the experimental plan and design.
00:58 - 01:06: And for this, we’re going to talk about materials, the controls of our experiment, the reagents
01:06 - 01:13: that are so important and it’s important because we need them to be ready for the whole experiment,
01:13 - 01:20: the color compensation that is important for our compensation, and the multicolor design.
01:20 - 01:29: So once we have a multicolor experiment, it’s important and it’s very related to our compensation.
01:29 - 01:36: Once we have our experimental plan and design done, we’re going to be able to run our experiment
01:36 - 01:41: at flow cytometry, and then in the end, we can do the data analysis.
01:41 - 01:44: So this is our plan for today.
01:44 - 01:48: Let’s start with the experimental design.
01:48 - 01:54: So for experimental design, the important thing, the first step is, what’s your question?
01:54 - 01:55: What’s your aim?
01:55 - 01:59: What do you need to be answered with this type of experiment?
01:59 - 02:09: For this, we have to remember, if this is going to be the first thing, this has to be
02:09 - 02:20: very important because we have to remember if we have equipment able to answer your question.
02:20 - 02:26: So we have to pay attention to the characteristics of the equipment, if this is going to be the
02:26 - 02:31: better way to answer our question.
02:31 - 02:33: The third point is reagents.
02:33 - 02:38: We have to remember that we need certain types of reagents to run this experiment.
02:38 - 02:46: For this, it’s important to have the same type of reagent from the beginning to the end.
02:46 - 02:52: You can move only, of course, if your experiment is not running well, but it’s very important
02:52 - 03:00: to have the same type of company providing the reagent to you, the same lot.
03:00 - 03:07: Everything has to be exactly the same just to avoid any difficulties, any differences
03:07 - 03:09: in your experiment.
03:09 - 03:11: The other thing is material.
03:11 - 03:15: So material means what type of sample you’re going to use.
03:15 - 03:23: So if you’re going to use cells from blood or from tissue, so these can be different
03:23 - 03:26: in one experiment to the other.
03:26 - 03:30: The other point for experimental design, it’s compensation.
03:30 - 03:36: This is a very important point here, and I’m going to show you later on after we have our
03:36 - 03:43: material with our reagents, how we have to think about compensation for an experiment.
03:43 - 03:46: The other point is fixation.
03:46 - 03:52: It’s important because if we don’t have a chance to run our experiment on the same day,
03:52 - 03:59: we can fix our material and run later on, but this can bring you some differences.
03:59 - 04:03: So we can discuss a little bit about this later on.
04:03 - 04:04: And about permeabilization.
04:04 - 04:13: So permeabilization, we have to think in a way if we have to use some compensation first,
04:13 - 04:16: and then we can have an intracellular staining.
04:16 - 04:19: So for intracellular staining, we have to permeabilize our cells.
04:19 - 04:26: This is very important, and this can bring you some differences as well in the surface
04:26 - 04:27: staining.
04:27 - 04:34: So we have to think about all these points here related to experimental design, just
04:34 - 04:40: to remember that we have to be aware of all this.
04:40 - 04:47: Once we have our experimental design there, we have to plan our experiment.
04:47 - 04:48: How are we going to do this?
04:48 - 04:54: First thing is to remember about quality control, what this means.
04:54 - 04:56: First it’s about materials.
04:56 - 04:59: We can have different materials to run our experiments.
04:59 - 05:06: We can have cells like cultured cells, cells from whole blood, or cells from tissue.
05:06 - 05:11: Each one of those options here can give us differences.
05:11 - 05:17: So just to keep in mind that we have to have good material to run our experiment.
05:17 - 05:19: The other thing is reagents.
05:19 - 05:24: As I told you, it’s very important to have the same type of reagents from the beginning
05:24 - 05:26: to the end of your experiment.
05:26 - 05:35: So you can just take a look at the lot number, expiration date, pay attention to your buffer
05:35 - 05:41: if they are exactly the same and you have enough for your whole experiment.
05:41 - 05:43: The other thing is, for example, PBS.
05:43 - 05:50: PBS we can have from different companies, so we can have those different reagents.
05:50 - 05:55: It’s going to have different lot numbers, different expiration dates, pay attention to that.
05:55 - 06:04: So this is very important that you have exactly the same type of reagents from the beginning to the end.
06:04 - 06:06: How about antibody titration?
06:06 - 06:08: Why titrate the antibody?
06:08 - 06:09: It’s so important.
06:09 - 06:16: Of course, we have a specific amount of antibody to use.
06:16 - 06:22: We have always an amount that it’s the perfect, the correct amount.
06:22 - 06:30: But sometimes, depending on the differences of your materials in your experiment, the source of the cells,
06:30 - 06:36: you can play the game with different amounts of antibody that you’re going to use.
06:36 - 06:46: For this, we have to titrate our antibodies to see exactly the correct amount to use for each experiment.
06:46 - 06:49: The other point is fixation and permeabilization.
06:49 - 06:55: As I told you, if we are not able to run on the same day, we can fix the cells.
06:55 - 07:00: You have to permeabilize your cells if you’re going to run an intracellular staining.
07:00 - 07:05: So for this, it’s very important to pay attention to quality control.
07:05 - 07:11: Why? Because we have different ways to fix and permeabilize cells.
07:11 - 07:14: And these can give you differences in your experiment.
07:14 - 07:21: If you start doing a fixation in one way and it goes well, do exactly the same way until the end.
07:21 - 07:23: And conjugation.
07:23 - 07:25: So this is another important thing.
07:25 - 07:30: If we’re going to use conjugated antibodies in your experiment or not.
07:30 - 07:40: So just to remember all these steps here, it’s going to give you the quality control for your experimental plan.
07:40 - 07:48: And once you have your experimental design and plan, then you’re going to start your experiment.
07:48 - 07:49: How are we going to do this?
07:49 - 07:54: So for your experiment, we’re going to need some samples.
07:54 - 07:57: First one, it’s unstained sample.
07:57 - 07:59: It’s the cells only by themselves.
7:59 - 08:01: No staining at all.
08:01 - 08:05: It’s going to be your baseline.
08:05 - 08:07: Positive, it’s your sample.
08:07 - 08:09: Your positive control.
08:09 - 08:13: And your negative control for what you were looking for.
08:13 - 08:16: Then we’re going to have isotype control.
08:16 - 08:18: This is very important.
08:18 - 08:24: Then we’re going to be able to identify exactly what we are looking for here.
08:24 - 08:26: Benchmarkers as needed.
08:26 - 08:31: If you need to make sure if your antibody is working well.
08:31 - 08:36: If you want a double proof, you can use benchmarkers in your experiments.
08:36 - 08:38: FMO, what does this mean?
08:38 - 08:41: This means fluorescence minus one.
08:41 - 08:43: This is part of the compensation.
08:43 - 08:50: And this means once you have a multicolor compensation, you can use FMOs.
08:50 - 08:56: It’s going to be if you have four colors for your compensation for your experiment,
08:56 - 09:00: always you’re going to have one tube with three colors.
09:00 - 09:02: That means minus one.
09:02 - 09:03: And this is going to be the next tube.
09:03 - 09:07: It’s going to be another three colors minus the other one.
09:07 - 09:09: So it’s always minus one.
09:09 - 09:12: This is going to give you the right compensation for your samples.
09:12 - 09:15: And then the experimental sample.
09:15 - 09:16: It’s your aim. 0
9:16 - 09:17: It’s your question.
09:17 - 09:21: It’s your tube that you want to answer.
09:21 - 09:24: We have compensations as I told you.
09:24 - 09:29: So for compensations or controls, we have unlabeled tube.
09:29 - 09:32: We can have a secondary antibody as a control.
09:32 - 09:36: We can have isotype control.
09:36 - 09:39: We can have benchmarkers if it’s useful.
09:39 - 09:43: And FMOs if we need it.
09:43 - 09:46: Cell fixation, it’s important.
09:46 - 09:51: Because this is going to give us differences in our experiment.
09:51 - 09:56: And if we are not able to run on the same day, we can fix the cells.
09:56 - 10:03: Permeabilization, it’s necessary once you need to use intracellular stain.
10:03 - 10:11: So once we have controls, cell fixation and permeabilization done in your plan
10:11 - 10:15: to do your experiment, you are ready to go.
10:15 - 10:19: Why it’s important, the configuration of the experiment.
10:19 - 10:21: So we have different materials.
10:21 - 10:27: Peripheral blood, cells from the skin and cells from culture.
10:27 - 10:31: And we have those cells in suspension.
10:31 - 10:35: Those cells will receive the fluorescence antibodies.
10:35 - 10:39: And those antibodies will be on the surface of the cells.
10:39 - 10:46: That is going to be the one they stain to go under the ground of the experiment.
10:46 - 10:48: This is very important.
10:48 - 10:53: So once we have our antibodies attached to the fluorophores,
10:53 - 11:01: this is going to give us a wavelength that is going to be important information for our experiment.
11:01 - 11:06: And this, we know that fluorophores are molecules that absorb energy
11:06 - 11:08: and are going to re-emit energy.
11:08 - 11:15: So we have the cell that’s going to be under your experimental design.
11:15 - 11:18: It’s there, it’s stained already with your fluorophore.
11:18 - 11:23: That is going to be identified by the equipment
11:23 - 11:26: to give you the information in the end about the wavelength.
11:26 - 11:28: And why is this important?
11:28 - 11:30: Because of the color compensation.
11:30 - 11:34: So we have different colors here in this chart
11:34 - 11:38: that is going to give us exactly what we need to know
11:38 - 11:44: once we are choosing our different colors for our color compensation in our experiment.
11:44 - 11:47: So we have Pacific Blue, for example,
11:47 - 11:55: doesn’t go in any way across the information with Alexa Fluor 647, for example.
11:55 - 11:57: So this is very important.
11:57 - 12:01: Once I choose Pacific Blue as one of the types of my antibody
12:01 - 12:04: and the other one is going to be Alexa 647,
12:04 - 12:08: this is going to be perfect because I will never have any problem
12:08 - 12:11: during my compensation because they are not overlapping.
12:11 - 12:15: So this is very important and this is why for compensation,
12:15 - 12:24: we have to learn about all our fluorophores that we discussed before,
12:24 - 12:27: especially in the first part of this webinar.
12:27 - 12:29: For multicolor compensation.
12:29 - 12:32: So once we know that compensation means
12:32 - 12:38: that we have to keep the wavelength apart from each other for each antibody,
12:38 - 12:42: we have to think about multicolor compensation.
12:42 - 12:48: Because we have compensation controls must match exactly the same fluorophore
12:48 - 12:51: with the experimental tubes that we got.
12:51 - 12:56: Compensation should be done once statistically and not by eye.
12:56 - 12:59: Second thing that is very important.
12:59 - 13:04: You have compensation beads commercially available that you can use
13:04 - 13:07: or you can do your compensation,
13:07 - 13:10: the single color compensation with your own cells
13:10 - 13:12: that you are using in your experiment.
13:12 - 13:15: So you have these two options.
13:15 - 13:16: Third thing and very important.
13:16 - 13:22: Once compensation has been set, don’t move, don’t adjust.
13:22 - 13:25: You cannot move anyway.
13:25 - 13:27: And the most important thing, the FMOs,
13:27 - 13:30: as I told you that is fluorescence minus one.
13:30 - 13:34: So this is very important because we’re going to have,
13:34 - 13:36: as I told you, for multicolor compensation,
13:36 - 13:40: we’re always going to have one tube minus one color.
13:40 - 13:44: So this is going to give you exactly what you want to see
13:44 - 13:49: for each color in each antibody.
13:49 - 13:54: So once we have our cells compensated already,
13:54 - 13:57: we can think if we need to fix those cells.
13:57 - 14:00: Because if we are not able to run the experiment on the same day,
14:00 - 14:02: we have to fix them.
14:02 - 14:04: We have here two options of fixation.
14:04 - 14:09: So paraformaldehyde keeps the cell morphology the same.
14:09 - 14:10: This is very good.
14:10 - 14:13: The other option is methanol ethanol.
14:13 - 14:17: This can change the morphology of the cells
14:17 - 14:20: and has to be performed at minus 20 degrees.
14:20 - 14:27: But this permeabilization allows the cells greater access to antigen sites.
14:27 - 14:31: So we just have to balance which option is the best one
14:31 - 14:34: once we decide to do fixation,
14:34 - 14:38: once we are not allowed to run on the same day our experiment.
14:38 - 14:41: The last thing is about permeabilization.
14:41 - 14:43: So permeabilization is very important
14:43 - 14:48: once you need to stain our cells intracellularly.
14:48 - 14:51: So this is very important.
14:51 - 14:55: The most appropriate permeabilization method possible,
14:55 - 14:58: we have tons of different ways to do it.
14:58 - 15:00: Just choose the best one.
15:00 - 15:03: Insufficient permeabilization, what does this mean,
15:03 - 15:08: means that you are not going to be able sometimes to stain inside of the cells.
15:08 - 15:12: And the other thing, once it’s not sufficient,
15:12 - 15:16: we can have high background levels in the antibody
15:16 - 15:20: that is washed out completely or is not sufficient.
15:20 - 15:25: So we have to think about this once we decide to do an intracellular stain.
15:25 - 15:30: We can consider, of course, the size of the fluorophore that you are using,
15:30 - 15:35: because if they are too large, we need big pores in the cells.
15:35 - 15:40: And if we have, the other thing, way to consider is the protein target.
15:40 - 15:46: This is a small protein may leak out of your cell because you have big pores.
15:46 - 15:49: So just to remember, if you need to permeabilize the cells,
15:49 - 15:54: you have to remember those little tricks.
15:54 - 16:02: Now we finish this first part that is about the design and the plan of your experiment.
16:02 - 16:07: We’re just going to the other part that is to run your experiment.
16:07 - 16:09: How are we going to do that?
16:09 - 16:14: So first thing to remember, it’s the configuration of your flow cytometer.
16:14 - 16:17: Because why is this important?
16:17 - 16:22: Because we need to remember that each equipment has different characteristics,
16:22 - 16:28: different numbers of lasers, different colors that they are able to capture the light.
16:28 - 16:35: So we have to remember this not to just have a spare experiment,
16:35 - 16:37: like an experiment that is not going to go well,
16:37 - 16:44: because we just choose a color that it’s not able to be read by that equipment.
16:44 - 16:48: We have to remember that we have to have single color tubes.
16:48 - 16:53: So each tube receives only one color into the cells.
16:53 - 16:56: And this is going to give us our compensation.
16:56 - 16:59: It’s going to be our baseline for each color.
16:59 - 17:05: Then we have isotype control, that is going to give us exactly the way
17:05 - 17:12: that we can differentiate the color that we are reading our antibody,
17:12 - 17:15: the question that you are asking, and the isotype,
17:15 - 17:19: that is not exactly what you’re asking.
17:19 - 17:22: FMOs, as I told you, it’s fluorescence minus one.
17:22 - 17:27: So we always have to have those once you have multicolor experimental.
17:27 - 17:30: And the other thing is your experimental sample.
17:30 - 17:36: So once you have all this ready to go, you are running your experiment.
17:36 - 17:39: And we’re going to go for each one right now.
17:39 - 17:45: Remember, flow cytometry has different lasers and filters in your configuration.
17:45 - 17:49: Here, it’s just an example as we have these three different lasers here.
17:49 - 17:54: And we have, we’re going to be able to read those wavelengths, those colors.
17:54 - 18:00: This means that I cannot use others different than those,
18:00 - 18:05: because it’s going to be a waste of money and a waste of antibodies and a waste of materials.
18:05 - 18:08: It’s not going to be able to read those.
18:10 - 18:11: So histogram.
18:11 - 18:19: Histogram is a way, one of the ways that we can just represent your data.
18:19 - 18:24: This means, for example, here, lots of events in a flow fluorescence,
18:24 - 18:27: in a low fluorescence intensity.
18:27 - 18:33: So this means that we have tons of events in this peak, but the fluorescence is low.
18:33 - 18:41: And here we can see in the other peak that we have lots of events in the high fluorescence intensity.
18:41 - 18:44: We can see the difference here in the histogram.
18:44 - 18:45: Why is this important?
18:45 - 18:53: Because this is related exactly to our compensation, as we can see the single colors.
18:53 - 18:56: So here we have the multicolor flow cytometry.
18:56 - 19:04: We have four different fluorescence here, Pacific Blue, PE, APC, and FITC.
19:04 - 19:12: As we can see here, the peaks, the peak with the high fluorescence and the lower one.
19:12 - 19:20: And those here, it’s just showing us where exactly we can see the FITC.
19:20 - 19:23: The APC, it’s a little bit lower.
19:23 - 19:26: The PE, it’s higher.
19:26 - 19:29: And the Pacific Blue, it’s 10 to the fourth.
19:29 - 19:38: So we have a way to see that this high peak is exactly the peak for the color that I’m looking for.
19:38 - 19:42: And the low peak is the color that I’m not looking for.
19:42 - 19:49: So those were done in beads, but you can use cells as well.
19:49 - 19:50: Isotype control.
19:50 - 19:56: So we have here represented in the black line, in the black peak, the negative control.
19:56 - 20:01: The isotype control peak is just in the first block.
20:01 - 20:10: The second one, the orange peak, is the one for the positive cell population that is stained for CD4.
20:10 - 20:15: So we can see the isotype in black, and in orange, the CD4 antibody.
20:15 - 20:27: So this means that you have a perfect shift between the CD4 and the isotype that you were looking for in this experiment.
20:27 - 20:34: Why is it so important once we have done our planned experiment and we have our design,
20:34 - 20:43: and we just run our samples and our compensations, then we go for the next step, the gating strategy.
20:43 - 20:51: This is going to just remind us that here we have FSC and SSC.
20:51 - 20:56: This is related to the granularity of the cell and the size of the cell.
20:56 - 21:01: And we have these three different populations there.
21:01 - 21:08: In this second plot here, we can see the live cells, the apoptotic cells, and the dead cells,
21:08 - 21:14: based on two different stains, 7AAD and Nxin5.
21:14 - 21:21: So we can have a lot of different information based on just two different fluorophores here.
21:24 - 21:33: Here, we have a lot of different colors, but it’s amazing how we can identify different groups of cells,
21:33 - 21:37: different sets of cells, based on the antibodies that we are looking.
21:38 - 21:44: So the first plot, as we can see in the FSC and SSC, we can have the lymphocytes.
21:44 - 21:51: The lymphocytes then were gated based on CD3, that is the T cells.
21:51 - 21:59: So once we have the T cells, the positive for CD3, we just have these other gates here.
21:59 - 22:06: From the T cells, we have the CD3 positive and the CD4 positive here.
22:06 - 22:11: For both positive cells, we have the T helper cells.
22:11 - 22:17: And we can still have the CD3 in FITC and the CD8 APC size 7.
22:17 - 22:21: And both positive, they are the T cytotoxic cells.
22:21 - 22:26: So those cells are completely different from the T helper cells.
22:26 - 22:33: And we have, at the same time, CD3 positive and CD3 negative.
22:33 - 22:40: And we still can have CD16 and CD56 as PE color.
22:40 - 22:45: So we have the NK cells; they are not CD3 positive.
22:45 - 22:50: They are only CD16 and CD56 positive cells.
22:50 - 22:56: And we still have here, on the other side, CD3 positive and CD3 negative.
22:56 - 22:59: And we have the CD19 APC.
22:59 - 23:05: They are positive for CD19, but they are not positive for CD3.
23:05 - 23:08: So these, those are the B cells.
23:08 - 23:15: So we have here different colors, different stainings, and of course, different sets of cells.
23:18 - 23:24: Here I’m showing data that we are just showing in a histogram plot.
23:24 - 23:33: This is just about our control, our isotype control, our negative peak of cells and positive cells.
23:33 - 23:36: So we can see on the red, our isotype control.
23:37 - 23:44: In blue, our negative sets of cells and orange, your positive cells.
23:44 - 23:51: So those peaks are very well separated and give us clear data here, as we can see.
23:51 - 23:53: And how about the data analysis?
23:53 - 24:00: So just remembering that we start looking for design and plan for our experiment.
24:00 - 24:06: Then we pass through how to run an experiment where we have to remember about it.
24:06 - 24:09: And then now we’re going to start with the data analysis.
24:09 - 24:13: Once you have your experiment run, how are you going to do your data analysis?
24:14 - 24:18: First thing to remember that the data analysis software has a range of tools.
24:18 - 24:24: That it’s able to use and visualize data in a variety of different ways.
24:24 - 24:31: So for this, you just have to remember, again, your first thing, what’s your question?
24:31 - 24:36: Then based on this, you can choose different ways to analyze your data and to show.
24:37 - 24:40: So here we have the first thing is the histogram.
24:40 - 24:45: Each histogram is just a single measurement parameter.
24:45 - 24:47: Single measurement parameter.
24:47 - 24:50: So we just have to show one single information.
24:51 - 24:56: If you have more than one information to show, histogram is not your option.
24:57 - 24:59: Then we have overlay histograms.
25:00 - 25:03: Those continue exactly.
25:03 - 25:10: We have just one parameter, but we can have positive and negative cells into the same
25:10 - 25:11: parameter.
25:11 - 25:17: So you can use this overlay histogram, but still it’s only one parameter.
25:17 - 25:21: If you have more than one, this is still not an option.
25:22 - 25:24: Other option is dot plots.
25:24 - 25:28: So dot plots, you can have two measurement parameters there.
25:28 - 25:32: So these start to be your option.
25:32 - 25:37: So here we can use the dot plots with the two parameters.
25:37 - 25:42: We can use two different parameters here to take a look at your data.
25:42 - 25:45: Other options are density plots.
25:45 - 25:49: Density plots, the cells are just counted as density.
25:49 - 25:54: So it’s just the density, the denser populations of cells.
25:54 - 25:56: It’s a little bit different because it’s three dimensional.
25:57 - 26:01: It’s not exactly the way that we are looking in the dot plots.
26:02 - 26:03: The contour plots.
26:03 - 26:12: Contour plots is another option to use that you can use for two different parameters here.
26:12 - 26:17: But this is very similar to the dot plots, but it’s lines here as a map.
26:18 - 26:19: So it’s another way to use.
26:21 - 26:23: And we’re going to have the gates.
26:23 - 26:25: Why the gates are so important?
26:25 - 26:30: Because the gates are going to allow you to exactly visualize which population of cells
26:30 - 26:35: are you looking for or which population you are interested in.
26:36 - 26:41: So you can have different gates here in each population.
26:42 - 26:47: Or you can have different forms for those sets of cells.
26:47 - 26:49: Or you can have regions.
26:49 - 26:53: Regions are not the same thing as gates.
26:53 - 26:58: So regions are going to give you a three dimensional appearance.
26:58 - 27:03: And it’s going to give you the percentage that you are looking for.
27:03 - 27:05: The percentage of cells.
27:05 - 27:07: So this is a little bit different.
27:08 - 27:09: And the quadrants.
27:09 - 27:10: What do the quadrants mean?
27:10 - 27:16: Means that you can divide in two dimensional plots into four areas.
27:16 - 27:21: This is going to give you very well separated populations of cells.
27:21 - 27:24: But this is defined by the user.
27:24 - 27:29: So you have to be very aware once you draw this.
27:30 - 27:34: Once I just showed you all this.
27:34 - 27:36: So now I’m ready for your questions.
27:36 - 27:39: You can email me your questions.
27:39 - 27:42: I will be very happy to answer all of them.
27:42 - 27:45: And it was my pleasure to be here with you.
27:47 - 27:54: Thank you so much.

About the presenter:

Sara is an Immunologist with expertise in multi-color flow cytometry and data analysis. During her master's and Ph.D. degrees at the Federal University of Bahia (Brazil), she researched human infectious diseases, such as leprosy and leishmaniasis. Her subsequent research focus has included coordinating and conducting flow cytometric experiments into the development of new potential therapeutics and immune system functions in disease. Sara is the Flow Cytometry Scientist at Abcam working in the New Product Development - Characterization team, and has published 26 papers in peer-reviewed journals.

Flowing in flow cytometry

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