Hi my name is Cecilia Lasser and I'm from the University of Gothenburg in Sweden. Welcome to this second part on Differential Centrifugation. This part is going to focus a bit more on calculations and I promise you it Is much more fun than it sounds like. Okay, so this is what we'll focus on in this second part of differential centrifugation. What is the clearing factor, the k factor? And how is this connecting to the pelleting time, the time needed to a pellet something. We will also look at the rotor conversions formula. What do you need to know if you switching from one rotor another rotor. And in the end I will mention some of the things that effects the EV yield and the purity. So when you're reading an article on extracellular vesicles and perhaps on exosomes, it's quite common to see something like this in the methods section. An ultracentrifugation step at 100,000 g for 70 minutes was performed to obtain exosomes and then you might ask yourself. Okay, where is the 100,000 g? Where is this calculated? Is it in the minimum? Or at the maximum radius of where the tube is located, where your sample is. So we're not going to look or talk about the formula in detail but we can see that the radius is part of this formula. And if we use the type 70 Ti rotor as an example, we can see that we need to pick one of these radius. And if we start with the maximum one, which is almost 92 mm and we use the calculations as you can see above. Then to get 100,000 g, down here in the maximum radius, we will put in 31,170 RPM into the instrument. Because that is what we put in to get this g. But what happen if we use the average radius instead? Because that only 65.7 millimeter then to get 100,000g at this point we will need to put in 36,865 rpm to the instrument. So this is actually different and this is just within one rotor but for different rotors of course they have different radius. So this is something to consider. If we're done looking at the clearing factor, the K-factor, this means the relative pelleting efficiency of a rotor at maximum speed. And again, we're not going to go into detail on the formula but we can see that the difference between the maximum and the minimum radius is included and also add the RPM. Simplified you can say that, the K- factor the lower, the close at to zero, the better pelleting efficiency it has. So if we look at some of the common rotors used we can see that the type 70 TI rotor on the top at its maximum speed it will have a k factor of 44. If we look at the type 45 TI rotor, it has a k factor 133. And for the swinging buckets 32 Ti rotor, it will have a k factor of 204. So this swinging bucket rotor is less efficient in piloting vesicles and particles. So then how is this k factor effecting the pelleting time. So T is the centrifugation time you need in hours, k is the k factor of the rotor, and s is the sedimentation coefficients that we talked about in the first part of differentials centrifugation So let's say we have researcher A. They have worked for a while on extracellular vesicles. They have a protocol that they use, they have probably published something. And researcher B is you in the field and is emailing researcher A and saying, hey, I'm interesting in exosomes. I'm going to start working on extracellular vesicles and could you send me your protocol? Sure, say researcher A, here it is, you have the time, you have the speed, you have everything. But for researcher B realized when it gets the protocol is that. Okay, researcher A have rotor 1. And when this person goes down in the lab and check they have a different rotor, rotor two and this is usually how it is. You have a certain type of rotors in the lab and those are the ones that you can use. New rotors are quite expensive to buy and you will just have to make it work with what you have. If you are using a similar body fluid or similar cells the sedimentation coefficient will be similar. As I said last time in the first part is that the sedimentation coefficient depends on the size and the shape of the vesicle that you're going to pellet. And the viscosity of the media that, the fluid that the vesicles are moving through. And if these are similar, you can just take the s out of the equation and now you have the time for the rotor 1 and the k factor time. And k factor for rotor 2, and you can now make your calculations on what you need to do with the pelleting time for your rotor. If you don't like to do the calculations your self, there are web pages where you can get help doing it If you have Beckman Coulter rotor and centrifuge, you can go to their webpages. But of course there will be similar things on other company webpages as well, but I have used this as an example, so you select rotor 1. In this example, I have chosen the type 70 Ti. You enter the run speed that you usually use. You enter the time that you are spinning. Then you select rotor 2, and in this case it would be the type 45 Ti rotor, you have done your calculations on hundred thousand G at the same spot as the other person or for the other rotor. And you now have RPM value that you can enter. And when you press calculate, it will tell you that you actually need to run this rotor for 94 minutes to be as effective as the type 70 TI rotor it is on in 70 minutes. And our group published some work in the genre of exo cellular vesicles in 2014. And in there we actually included a table just to show that if we used the type 70 TI rotor the standard that we compare with, it means that for the swinging bucket rotors, we need to centrifuge longer. As you can see here, that would be 144 minutes because they have a lower k factor while for example the TLA 100.3 rotor. Which is at the bottom have a very low k factors actually you do not need to spin that long time to get the same pelletting efficiency. So this is important to remember if you want to compare yield or something with somebody else using a different rotor stat. You should not just copy their protocols if you don't have the same rotor in your lab and we can see it here. We have taken a type 70 Ti fixed angle rotor and compared with the swinging bucket 32 Ti rotor and we are just running them for 70 minutes, both of them. And no calculations, no changes. And we can see with the RNA yield that it was significantly higher For the fixed angle rotor, which was expected since it has the lower k factor. But we did calculations as I showed you previously and we then solved that okay, we need to do 114 minutes to get equal pelleting efficiency. And now we can see that there's no significant different between the RNA yield for the fixed angle and swinging bracket rotors when we do these calculations. Similar thing is can be said for Partially Filled Tubes which was shown by Jeppesen et al, in Journal of Extracellular Vesicles in 2014. And also for Partially Filled Tubes there are calculations. And when this where use and the time was adjusted it was shown that the yield were singular. So by prolonging your centrifugation, you will obviously increase your yield. So that would be quite nice to do if you have a low yield. And we can see here in this paper from 2014 that we tested to go from 70 minutes all the way up to 37 hours. And we looked at RNA yield in our palleted vesicles and we can see that it was increasing but if you remember this picture from the first part of this lecture. You can see that for each belonging time the centrifugation coefficients get lower so at 37 hours, you have a very very low S value which means you are pelleting soluble proteins as well. So you should not just prolong your centrifugation without noticing that the purity of your sample will go down. I wouldn't recommend 37 hours but maybe two and a half or four hours would be interesting. But you would probably have to combine that with either density gradient or size exclusion chromatography, that. Will be talked about later in this course to remove the soluble proteins from the vesicle fraction. So, in the paper from Jeppesen they also showed that the optimum G force for different cell lines might not be the same. So what we can see here is the ratio between particles per microgram of proteins. So you want as many particles per protein. This is used as it gets lower it means that you have start pelleting soluble proteins. And they could actually see that it was different for these two cell lines. One had the optimum at 67,000 g, and one had the optimum at 100 1000 Gs. So, that could be something to consider for your cell. That is could actually not be the same and of course, the viscosity affects the EV yield. The higher the viscosity, the harder it will be for the vesicle to get pelted to move through the media. And plasma and serum have higher viscosity than conditional media and PBS. And this greater viscosity lead to a lower sedimentation efficiency. So that means when you're working with this type of samples or another source of the extracellular vesicles that are very viscous, like saliva or sputum or something like that. You need to dilute them in PBS, so the conclusion of this second module of the differential centrifugation is that we have talked about k factor. And this is the relative pelleting efficiency of a rotor and the lower the k factor the better the pelleting efficiency. When following a published protocol, you should check which rotor was used and where are the g-forces was calculated. And so you can adjust and recalculate for your rotor which time you would need to have a similar efficiency in pelleting your basic calls of interests. You need to use proper calculations when you're using new rotors. Those are available both in In the Jefferson paper but also in the paper from 2014 in. But they're also easily found on different web pages several factors can effect the EV yield and purity. Which rotor you use, the degree of filled tubes, centrifugation time, the speed you choose, and the viscosity. So when you are going to start up your isolation of excess. Don't just copy somebody else protocol, without adjusting it for the sample that you have, and the equipment that you have in your lab. Thank you for listening to this lecture.