Indução e Monitoramento de hipersensibilidade tardia-Type Adoptive em Ratos

I am 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 in the next few days I'm going to show you the procedure of inducing and measuring adaptive DTH in Louis rats.

So this procedure takes five days and what I'm going to show you is how to activate antigen specific T cells, how to then two days later, inject them intitally into naive rats. Then two days later, we will challenge the rats in the ears with the antigen for which those T cells are specific. And then a day later we are going to measure the degree of inflammation in those rats.

To get serum for my tissue culture, I need to get the blood. So for that I do a cardiac puncture. I put my left index on the solar plexus and I reach to the heart through the ribs, between the ribs with my needle and I aspirate as much blood as possible.

And from one draw, depending on the day and the efficiency, we can get between two and 10 mils of blood from a given rat. And the reason why we need serum in the culture, we want to grow cells which are Louis rat cells in serum, that's syngeneic. So those will be Louis rat serum.

I then transfer the blood into a tube, but I'm careful to remove the se, the needle from the syringe so that I don't have hemolysis because I want to use my serum for my tissue culture. Later on. Now I'm going to remove the thymus since the heart has stopped beating and the rat is dead.

So I first spray with 70%ethanol to make sure there's no fur flying around and going into my organ because I need it for tissue culture. And I use autoclaved instruments to do the dissection. So I first cut off the skin.

Once I've removed the skin, I carefully cut on each side of the thoracic cage to make sure I do not damage the thymus that's sitting just above the heart. And the ths can be seen here as a pale, almost white organ sitting just above the heart. So I remove it, making sure I take the whole organ, but I'm not too careful in taking it out cleanly because I'm going to clean it up in the hood later on.

And I simply place it in a tube containing PBS that I supplemented with penicillin and streptomycin. To avoid any bacterial growth, I am now going to make a single cell suspension of my cyte. And for that I'm going to use a 70 micron cell strainer.

And I'm going to use of course, all sterile equipment. I transfer the thymus into a Petri desh. And as you can see, it's not very clean.

The thymus is the whiteish organ and there are lymph nodes and blood clots on it. So I'm going to remove those. And I'm going to put about 10 mils of PBS supplemented with antibiotics in my cell screener to be ready to receive the thymus once it's cleaned.

So to clean it up, I simply remove all the contaminants from my thymus. Now my thymus is almost completely clean, so I'm going to pick it up with my tweezers and cut it in small pieces with my scissors above the cell trainer and put all the small pieces of thymus inside the cell trainer. And after I'm done with that, I'm going to use the plunger of a one mill syringe, which is ster of course, to make the single SU suspension by pressing the pieces of timers through the cell trainer.

And the single cells are going to come through and be in my PBS on the outside of the cell trainer. So now I've pressed my cell through the cell trainer and you can see that the PBS is not clear anymore. It's filled with my single cell suspension that I'm going to transfer to a 50 mil tube that I keep on ice.

And I'm going to repeat the procedure a couple of times by refilling my cell trainer with PBS pressing more with my syringe plunger transferring to my tube until I have between 30 and 40 mil of PBS in my tube. And this is what the cells strainer looks like once I'm done. Now that I have collected all of the cells, I'm going to spin them down for about 10 minutes At 1200 RPM.

Here are the cells that I took out of the centrifuge and the pellet is pretty large, which is expected from one time I'm now getting rid of the snet. So the pellet of course still stays at the bottom of the tube. And to make suspension more easy, I'm going to break the PT while it's still almost dry.

And for that I just tap it with my finger pretty hard until you see that the PT is completely disaggregated and we have, we are back to a single cell suspension. I'm now going to Resus suspend my cells in my sterile PBS and I'm adding five mil of PBS per timers. And since I only have one timers, I'm just adding five mils.

And this is the suspension I'm going to bring to the irrigator to origin. And the irrigation level I use is 30 degrees to make sure the cells are still alive but are going to die within a few hours. So they can still process the antigen, but they will not contaminate my culture.

If we take a look at the T cells that we want to activate, we see that they don't look healthy at all. Some have started dying. They have strange shapes, they're not found anymore.

So those cells are ready to be activated. They're actually desperate for antigen after the time sites. It's extremely important to wash them quickly because some of them will have died and will release toxic compounds in the solution.

So I add, I complete the tube up to 50 mils with my PBS solution. And at the same time I'm going to transfer the T-cell that are waiting for stimulation. I'm going to transfer them into another 50 mil tube so that I can send refuge both tubes at the same time.

So here's the blood I collected a little while ago, and I left it for 10 minutes at 37 degrees and then I put it on on ice. And as you can see, it has completely clotted. So I need the serum out of that blood.

So to collect it in the first step using a sterile pasta pipette, I'm going to unstick the clot from the tube and I'm going to collect all the liquid which will contain the serum I'm interested into. And also some cells that were not trapped in the clot. And this liquid, I'm going to transfer to another tube during the transfer, sometimes small pieces of clot come along, but that's not so much of a problem because I anyway need to remove the cells that are still in that liquid.

So I'm going to centrifuge my small tube with the liquid in it. So that will remove the cells and any piece of clot that comes along, I'm now going to spin the liquid part of the blood just to separate the cells from the serum. So spinning for two minutes will be enough.

Now the serum has been spun, so I'm going to remove the serum from the cells. This is not a perfect example because the serum should be yellowish and this one is reddish showing that I had hemolysis. So this is not absolutely perfect, but I'm anyway going to show you how I filter it.

I use oh 0.2 micron syringe, adapted filter, and I'm going to filter my serum to make sure it's sterile. But since I had hemolysis, I will not use this patch of serum for my stimulation. I will use Louis rat serum that had frozen down during a previous experiment.

Here are the PTs of the T cells on the left and the antigen presenting cells on the right. I'm now going to get rid of the snat from both tubes. And as earlier, I'm going to break the pellets by tapping with my finger to make sure the cells are back to a single cell suspension.

And I'm going to do that for both set of cells, the T-cell and the antigen presenting cells. After that, I'm going to suspend them and I'm going to count them. I'm then going to mix 3 million of the T cells with 150 million of the antigen presenting cells, and I'm going to put in 10 centimeter diameter pet dishes.

And in a total volume of 10 mils, here are two tubes of serum. And the right is the one I prepared today and it's reddish because of hemolysis, whereas on the left is a tube that I prepared a few days ago and kept frozen. It's yellowish, it has no hemolysis, it's much better.

That's the one I'm going to use for my tissue culture today. I have enough cells to stimulate six Petri, so I'm going to put 30 mils of medium in the antigen presenting cells and 30 mils of the same medium in the T cells. And then what I'm going to do is in the antigen presenting cell tube, I'm going to add the antigen, which is of albumin.

So my stock is at one milligram per ml and I want a final concentration in my Petri dishes of 10 microgram per ml. And in the T-cell I'm going to add the Louis rat serum. And I'm going to add enough so that in my petri dishes in the end I have 1%rat serum.

I'm going to homogenize my suspension of antigen presenting cells and then I'm going to transfer five mils of this cell suspension into each of my Petri dishes. When you take out your suspension of antigen presenting cells, you'll notice always there's a little blob of fat and fibrous tissue. It's absolutely normal.

It's there in all our time of site preparations. It's nothing to worry about. So I just either get rid of it if I manage or otherwise I just leave it in the Petri dish.

It doesn't affect my cell culture at all. And once I've transferred all of the APCs into my Petri dishes, I'm going to repeat the procedure with the T-cell. So I'm going to put five mils of T-cell suspension in each of those Petri dishes.

Now all of my cells are in the Petri dishes and it's very important to mix them really well so that all the T-cell get a chance of being in contact with an antigen presenting cell. So for that, I pile up my pet dishes and I'm going to mix by doing invisible, drawing invisible eights with my Petri dishes. And I do that a few times to make sure all the cells are well mixed.

And once I've done all the mixing, I'm going to put those dishes in an incubator in a humid atmosphere at 37 degrees C and 5%CO2. I've been an incubator for 48 hours. The medium has slightly changed color, it's more orange now and the cells have been well activated.

So I check all the Petri dishes one by one under the microscope. So the very big cells are the activated T-cell and the very small ones are the dead antigen presenting cells. And the stimulation is really good because you can see that the T cells are huge.

And if I move the Petri dish around a little oops, you see on the left side here, now it's in the center, there's a big clump of cells and those are divid actively di dividing cells. So I'm now going to put all the cells into centrifuge tubes to be able to spin them down, count them, and then inject 10 million to rats in. So the stimulation was so good, I don't need all those T cells to inject to one rat.

So what I'm going to do is I'm going to use only cells from two petrides. So I'm going to spin them apart to inject 10 million to rat the remaining of the cells. I'm going to put in culture to freeze in a few days.

And the culture medium is the regular T-cell culture medium to which add T-cell growth factor and horse serum. Well, the cell T-cell are nonadherent, so I don't need to do anything much. I just make sure most of the cells come with the medium.

And then what I'll do at the end is I'll rinse all the petrol dishes with new medium to make sure I get all the cells out. 10 mill of media, the same one I used for the stimulation and then I think every pot dish, but the tcell have already come in the first time I took the medium. So the medium normally comes out pot here, it's not absolutely necessary, but since those cells are precious, I don't want to lose any.

So I'm going to spin them down. That will remove some of the dead cells and mostly start with that cells. And I want to give them a fresh medium.

So I'm Again going to spin that 1, 500 RPMs for about eight minutes. And I still leave the break on because I'm just paling the cells. So we just pan down both troops and in one I have the small pellets.

This comes from the two Petri dishes. And here's the pellet from the four Petri dishes. The pellets are huge because they don't contain only the activated T cells, but they also contain the data antigen presenting cells.

So now I'm going to empty the supernatant and in the cells I'm going to inject here I'm going to add one milliliter of medium, which is what I'm going to use to inject the right in. And the other cells I'm going to suspend in a hundred mils of culture medium with T-cell growth factor and horse serum. So those are the cells I'm going to inject.

And for these cells I'm going to put them in two plastics of 50 mil each. So in each flask I'm going to put 10%of T-cell growth factor, which is bone made. I also put 10%heating activated horse serum.

Then I'm going to add most of the medium I need. I don't pipe it up and down because T cells don't like being pipetted too much. So I just pipe them the minimum, absolutely necessary amounts they don't like being and I just put her finish and they're ready.

They're ready to go in the incubator, I check on them every day. I probably need To divide them after 24 hours, if not after 40, 48 hours for sure. But I'll check them every day.

They have a tendency to grow very fast in T-cell growth factor. So they need to be divided at least every 48 hours, probably sooner. I'll check also the color of the culture medium.

Now they're activated, so they're fully activated. That's the best time to inject them to the rat. And in two days we'll challenge the rat in the ear.

If we wait for the cells, if we wait a couple of days for the cells to go back to rest, they want home to the site of inflammation that fast. So now I'm going to put them in the incubator at 37 degrees centigrade with 5%CO2, I'm going to use a 25 gauge needle to inject the rat init. So now that the cells have been injected in ally, I'm going to leave the rat here and I'm going to challenge it in the ears in 48 hours.

So days ago I injected GFP labeled of of albumin specific T cells in to a lowest rat. And today I'm going to challenge the same rat in the ear. So in the right ear I'm going to inject of albumin.

And in the left ear I'm just going to inject some PBS. The dose of volin I'm injecting is 20 micrograms in 20 microliter. And the albumin I'm using is a mixture one to one of Texas red labeled albumin and unlabeled of albumin.

So this is the toilet on which I'm going to show you how I'm going to inject. So I have my left hand, I'm right-handed. So my left hand, I took the glove off to be able to put one finger under the ear and the other one above to stretch the skin so that I can see exactly where I am with the needle and go in between the two layers of skin in the air, which is very thin.

So for that, I use 27 gauge needles to have something extremely thin to go in there. So if I turn it around, you definitely see where the Texas red went and where the PBS went. So we are now 42 hours after challenge in the year, and I'm going to use a spring loaded micrometer to measure ear thickness.

So I'm going to measure both ears, the right ear that got challenged with the albumin and the left ear that wasn't challenged. And the difference in ear thickness will give us an idea of the intensity of the inflammation. So what I showed you here is the entire procedure to perform an adaptive DTH in rats.

So I showed you how to activate antigen specific T cells in vitro, how to transfer them adoptively intra to rats, and then how to challenge those rats and measure the degree of inflammation. We in the Chand lab use this procedure routinely to test the effect of potassium channel blockers in the inflammation in Rats.