Hello, I'm Christine Beaton. I'm an assistant researcher in George Chen's lab in the Department of Physiology and biophysics of the University of California at Irvine. And I'm going to show you today how to negatively select natural killer cells from human blood using the stem cell technologies cocktail kits.
The reason why we use this kit from stem cell technologies is that it gives us a very high purity of cells and the negative selection allows us to get cells that have not been bound to an antibody. So you don't have anything unexpected happening with those cells. So the way the tsep kit works is you have antibodies that are going to bind to all the cells you're not interested into in the blood.
So in my case, T cells, B cells, monocytes, and then you have antibodies that are going to bind to glycophorin A in red blood cells, what they did is they linked one antibody against a white blood cell to an antibody against a red blood cell. So what's going to happen is you're going to have forming of a complex with one unwanted white blood cell with multiple red blood cells and since red blood cells have a higher density than white blood cells, once you put your mixed cocktail containing blood and your antibodies onto a gradient, you are going to spin, the red blood cells are going to sink to the bottom of the tube and they're going to drag with them all the white blood cells you do not want. So on top of ingredient, what you're going to collect are your pure or almost pure natural killer cells that have not been bounced to an antibody at all.
So let's get started to isolate NK cells From a tube of human blood. So this is a freshly drawn blood on the sodium heparin tube. I'm going to use the Roset ep, human NK Cell enrichment cocktail as a gradient.
Later on in my experiment I'm going to use the 10 77 histo pack and this has a density of 1.077 and I've also prepared a bottle of PBS supplemented with 2%fetal calf seven. And I make sure that these components come up to home temperature before I use them Because I store them in the fridge. So I am going to take the whole tube of blood And put it in a 50 mil fcan tube.
So to, since it's a vacuum container, there's vacuum inside to open it without splashing, I localize the little notch on the lid on the cap and I lift it carefully to let air come in my tube. And when there's, once the the, the vacuum is broken in the tube, I can more easily take the cap out and then taking a 10 mil pipette, I'm going to pipet the entire tube of blood. So I expect there should be about 10 mils of blood.
Here I have 9.5 mils of blood and I'm going to put this whole 9 5 5 mils in this 50 mil falcon tube. And then the pipette with the blood in it goes directly in the biohazard bag, the tube, I always put some bleach in it, close it and then put it in the biohazard bag. So this is what the blood looks like before adding the antibody cocktail.
I'm now going to add the antibody cocktail to the blood since I had about 9.5 mil of blood and I need to add 50 microliters of cocktail per mil of blood, I'm going to add 450 microliters of the cocktail to my blood. And then I'm going to mix gently and almost immediately you are going to see the ting happen. As you can see now you see the ting.
So the cross leaking between the unwanted white blood cells and the red blood cells is happening right now. And I'm going to set up a timer for 20 minutes and let the tube sit like that in the Hood for 20 minutes. The blood has now incubated for 20 Minutes with the rosette sub cocktail.
So I'm going to dilute it one to one with the PBS plus fetal calf serum. And this mix I'm going to overlay on two 15 mils of histo pack 10 77. And after that I'm going to spin my ion in the centrifuge for 20 minutes.
So I have about 10 mils of blood plus antibody cocktail. So I'm going to add 10 mils of PBS containing 2%fetal cal serum. So that's a one-to-one dilution.
I'll have to mix gently because as you can see when I add the PBS to the blood, it doesn't mix immediately. So for that I just mix gently. So now I have a one-to-one mix of PBS with blood and this is what I'm going to add to my 15 mils of histo pack.
And I'm going to carefully overlay it to avoid mixing the blood with the histo pack. So for that, instead of using 25 M pipette that would get all the blood, I'm going to use a 10 10 mil pipet and go in several steps. So to overlay easily, I hold my histo pack tube almost horizontal.
I'm going to drop a little blood on the tube and bring it down to the histo pack. And then very slowly I'm going to layer the blood onto the histo pack. Once I have a good layer of blood, I can actually start piping a little faster and I can bring back slowly my tube to a vertical position.
Now I have overlaid my entire 20 mils of mix of blood with antibody cocktail and PBS on two 15 mils of histo pack. And if you can see at the junction of the tube, you start seeing slowly little aggregates of red blood cells sinking slowly into the gradient and we're going to centrifuge RET simply to make the process go faster. So within 20 minutes we will have all the red blood cells at the bottom of the tube.
And there is one very important point when doing a gt, you want to turn the brake off because breaking too fast might disrupt your ingredients. So we always switch the brake off and then I'm going to send my son RI for 20 minutes and let it do its job. The ingredient has now been spun for 20 minutes.
So we have the different layers of cells. We start at the bottom of the tube here. Here are all the red blood cells and the cells we're not interested into.
Above it there's a clear layer of gradient and above that there's a a yellowish layer of plasma. And the cells we're interested in two. The NK cells today are this thin white flu of cells between the gradient and the plasma layers.
So of course this layer is very, very thin because NK cells only represent a minority of the blood cells. Now I'm going to remove the layer of NK cells using a sterile past pipette. And this layer I'm going to put in a new empty 50 mil Falcon tube.
So I go to the layer and I pipe it out. Of course, there are so few cells. When you have a lot of cells, you can get the layer very cleanly when there is so few cells.
And as it's recommended on the notice of the manufacturer, they recommend that you take some of the gradient with the cells to avoid losing them. So I pipe it out as long as I do see some cells there. Once you've taken all the cells out, you should see a sharp contrast between the cleared regent and the yellowish plasma.
So I believe I have all my cells out now. So if you look at the gradient, you don't see anything white and fuzzy at the interface between the histo pack and the plasma. So now I've removed all the NK cells I could see from the gradient, placed them into a new 50 mil Falcon tube, and I immediately added some PBS supplemented with 2%fetal calf serum to the tube.
So I filled it up to 50 mil and I'm going to spin the cells down to pellet them. That will be the first wash. And I'm going to repeat the procedure a second time to wash the cells for a total of two times.
And after that, the cells will be ready for any procedure you want to do, either putting them in culture or using them for flow cytometry staining. And you should have at least 80%purity of NK cells, maybe more. I've had up to 98%purity with this procedure.
So today I've shown you a very rapid And reliable technique for isolating natural killer cells from human blood using the stem cell technologies TS kits. And of course if you want to isolate other cell types from either human rat or mouse, you can use any of the rather T cell isolation kits. They have some for T-cell, B cells, and even Subtypes of T-cells, for example.
