EAE와 쥐의 중추 신경계에서 Mononuclear 세포의 분리

Okay.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 Irvine. And today I'm going to show you how to isolate mononuclear cells from the central nervous system of rats.

So this procedure involves deeply anesthetizing rat to perform a cardiac perfusion so that we do not have any contaminating blood in the central nervous system. When we take it out, then we'll take out the brain and the spinal cord will make a single cell suspension using a cell strainer and then using a double layer gradient of perkel, we're going to isolate the mononuclear Cells. So let's get started.

I am now ready to do the cardiac perfusion, so I'm going To show all of the material I need for that. So the first thing we need is a Bel jar, and down here we've put paper towels soaked with volatile anesthetic. You can use halane or iso fluorine.

The next thing we need is something to hold the rat and something to collect the blood. So we have this makeshift installation to perfu the rats. You are going to use a 21 gauge needle, and we are going to peruse a saline solution gravity fed through an IV perfusion system.

If you do not have access to one of those systems, you can also fill several syringes with saline and perfuse them by hand. But this is much easier. And what we also need is a 10 mil syringe barrel in which we've put Kim wipes.

And I'm going to put the anesthetic, some of the anesthetic in it to put on the nose of the rat to make sure it stays deeply anize during the procedure. And then of course, you need instruments. So today I'm not going to do a sterile procedure because I'm not going to put the cells in culture.

So everything I'm using instrument wise are not sterile. But of course if you want to put your cells in culture, you may want to use sterile instruments. For the purposes of this, of this video, I'm going to use a rat that's actually dead.

So I can do the procedure slowly to show you, show it to you step by step. Of course, in a real procedure, you would need the rat, deeply anesthetized, but not dead. You would want the heart still beating.

So let's get started with this rat. So I placed the rat on, its back facing away from me, and I put the soaked team wipes over its nose. And then the first thing I'm going to do is I'm going to check that the rat is deeply an ized.

So you need to do that before each of those procedures. So I take my tweezers and I pinch the rat, and there's no reaction at all. If there's any reaction, you need to put the rat back in the belger to make sure it's deeply anesthetized.

Then I'm going to use 70%ethanol to soak the top half of the rat. And then I'm going to open the thoracic cavity. So first I'm going to cut the skin off and open it on the side.

Then I'm going to open the cavity itself. This mass here is the heart. Here you can see the right atrium.

That's what I'm going to cut to allow the blood to flow out. And then to perfuse the entire body, I need to insert my needle into the left ventricle. So I'm going to insert my needle right down here.

And once the needle is inserted, I'm going to open the IV tube to let the saline flow through, and I'm going to leave it for five to 10 minutes. If the perfusion is good, you are going to see a discoloration of the red pulse and red nose and rat eyes, of course, showing that the blood is leaving the rat body. So that means the perfusion should be good.

Here is the red after cardiac perfusion. So as you can see the nose, the ears and the eyes of this red are no longer pink or red. They're completely white, which shows that the perfusion worked well.

All or most Of the blood has left the rat spots. So now that the rat has been cardiac perfused, I need to cut its head off. I need to decapitate the rat.

And for that I'm going to use regular lab gear. So now I'm going to remove the brain from the rat. So going to soak all the fur with 70%ethanol just to avoid having fur flying all around.

And then I'm going to remove first the skin. And since I don't want to use the brain for histology, and I'm going to cut it in small pieces, I'm not terribly careful at taking it out cleanly. In one piece, I'm going to insert my scissors at the base of the skull where the brain connects the spinal cord right here.

And I'm going to cut the bone on each side and then I'm going to remove the top. And I'm going to do that again and again. So here is the brain completely exposed.

And as you can see, the perfusion worked well because the brain is completely clear. In a non perfused animal, you would see that the top layer of the brain would be completely covered with red capillaries, red blood capillaries, so you don't see them at all, which means the perfusion worked well. So now I'm going to scoop the brain out, making sure I don't leave the olfactory bulb behind, and I'm going to put it in a tube containing some ice called PBS.

So now still, without being very careful to keep it intact, I'm going to put the brain into the tube of PBS and I'm going to cut the optic nerves and then using a spatula, it's going to come out pretty easily. So now I'm going to remove the spinal cord from the same rat. And there again, since we're going to cut it in small pieces, I'm not going to be too careful about taking it out very Cleanly, as long as I get all of all of it out.

I first cut the skin all along the spinal Column and the spinal column is visible here. Then I'm going to remove the meat here so that the spinal column is exposed all along. And right here you can see the spinal cord.

So in a mouse, you can cut both ends and shoot PBS with the syringe and the spinal cord will come out in almost one piece. But in rats it's not that easy. I have to cut it open and scoop the spinal cord out.

So now I cut on both sides, and like I did for the brain, I cut and I remove the brain, the the bone so that I expose the spinal cord. And as for the brain, the spinal cord is completely white, which shows that the cardiac perfusion worked well. And I'm going to do that all along the spinal column.

And I go down to about the level of the right, the right hips, which is right here. And then I am just going to use to tweezers, and this is not very pretty, but I'm just going to pull out the spinal cord and put it into my tube with the brain. So now in my tube, I have both the brain and the spinal cord of the rat, and now I'm ready To prepare my single cell suspension.

So now I'm ready to Prepare a single cell suspension from my central nervous system. So what I'm going to need for that is 10 centimeter petri desh in it, I'm going to plus place 70 micron cell strainer. I'm also going to use the plunger of the one mill syringe, and I need tweezers and scissors.

So I'm going to put my sample into the cell strainer and make sure there's nothing left in the tube. Then I'm going to cut it in small pieces, both the brain and the spinal cord mixed up. So when everything is finally minced, I'm going to use the plunger of the oneill syringe and I'm going to press it through.

And you're going to see progressively the PBS outside the cell trainer is going to get cloudier as single cells go through. If I wanted my cell suspension ster, of course I would do that in a tissue culturehood and I wouldn't put my fingers in it. So now that the PBS is cloudy, I'm going to collect it first time and put it in a 50 mil tube.

But I'll also keep on eyes And I'm going to add some more PBS to my cell trainer, and I'm going to squash through. So since the CNS contains so much fat, it's, it's one of the organs that I find most difficult to prepare a cell suspension from using this technique. So I've pressed all my CNS through the cell trainer multiple times, and I've collected all the single cell suspension.

And here is the tube that I filled up to 50 mil with PBS containing my single cell suspension prepared from the CNS of the right. Now, I'm going to spin this tube down To prepare it for the gradient so the cells are finished spinning. We have a pellet now, so I'm going to discard the snet and I'm going to rest suspend my cells in 20 milliliters of perkel of 30%perkel in PBS.

And I'm going to overlay that onto 10 mils of 70%perkel in PBS to make migration. So I discard the SuperNet. I break the pallet to make sure the cells are in a single suspension and not in a clump in the pellet.

So now I have my suspension of CNS cells in single cell suspension. I'm going to suspend those cells in 20 mils of 30%perkel in PBS, and I'm going to overlay those 20 mils onto 10 mils of 70%perkel on PBS. And then I'm going to spin it down.

So now I'm going to take my 20 mils of CNS suspension, and I'm going to overlay it slowly onto the 70%perkel layer to make it easier. You can also add a dye like phenal red in one of the layers, but I'm not quite sure what it does to the cells if it does anything. So I try to avoid it.

You want to go slowly enough that the two layers don't mix, so you don't want any big turbulence in your lower layer. And as the top layer gets thicker, you can go faster. So now I have added my cells to the gradient.

So the clear layer down here is the 70%perkel layer, and the cloudy layer on top is a 30%perkel layer that contains my single cell suspension. And I'm going to now put this tube in the centrifuge for 20 minutes, and I'll be able to visualize the layer of monocle Cells if I worked well. So now we've done spinning the Gradient.

So as always, you spin a gradient, be very careful to switch the brake off so that it doesn't disrupt the gradient by slowing too fast. And so now we have our layers. We have on top of the gradient, a layer of fat that I'm going to remove to avoid contaminating my cell suspension.

And at the interface between the two layers of perkel 30 and 70%you see that the interface is not sharp, it's fuzzy, and that's where the cells are. Once I've removed the cells, you're going to see that the interface will be much sharper. So I'm just going to remove the major part of the fat on the top layer and just put it in my waist.

I don't need to remove all of it, but I want to remove enough so that it doesn't completely coat my pipette when I go down to get the monocle cells. And now what I'm going to do is using another transfer pipette, I'm going to go to the level of the interface and I'm going to harvest my cells. So now I've collected several mills at the interface.

So I probably have all the cells I could get there. The number of monocle cells in that CNS were really small. So now I have my mononuclear cells in the tube.

I'm going to fill that tube up to 50 mil with PBS, and I'm going to spin the cells down to pate them, and that's going to wash the perle away. And I recommend washing them at least a couple of time with a full tube of PBS to make sure you get rid of all the perle. And then the cells are ready For any procedure you want to do with them.

Today I've shown you how To isolate mononuclear cells from the central nervous system of rats. So of course, once you have those mononuclear cells, you can further purify them to isolate, for example, T lymphocytes outta the population. What I am going to do with those cells is to run flow cytometry assay to detect the cell populations that were in my brain.

And good luck With your cell isolation.