Hi, I am Christine Beaton. I work in George Chandy lab in the Department of Physiology and biophysics at UCI. Today I'm going to show you how to induce active DTH in rats.
So for that I'm going to prepare an emulsion of KLH, which is my antigen in France adjuvant. And then I'm going to inject this emulsion at the base of the tail of Lewis Rats. And this is going to induce immunization of the rats against KH.And in one week I'm going to challenge the rats with KH and measure the DTH reaction by swelling measurement of the swelling.
That's the site of challenge and we are using that in the lab to see the effect of PO channel blockers on inflammatory reactions such as DTH. So here is the incomplete trans adjuvant I'm going to use to make the emulsion. I could also use complete trans adjuvant.
Here are quartz of KLH, which is my antigen today, and I have a solution here at two milligrams per ml. In PBS, I'm going to use this five ml syringe to take out the incomplete trans adjuvant and transfer it into a 15 mil tube. I'm then going to put a tube on the vertex and while vortexing, I'm going to add my antigen drop by drop to the adjuvant.
And then I'm going to vortex for further five minutes. After that's done, I'm going to fill this five M glass syringe with my mixture and it has to be a glass syringe because the adjuvant has a tendency of eating plastic. And the five M syringes we found are the best for making the emulsion.
Smaller or bigger are not as good. And once I felt my syringe with the mixture, I'm going to use this metal bridge to link this glass syringe to another five mil glass syringe. And then I'm going to apply pressure on the syringes to have my mix go from one syringe to the other as fast as possible until it gets really hard until the, the emulsion gets really hard and then it's ready for injection.
So I'm going to put 2.5 mils of my adjuvant. So I just inject air to be able to get the adjuvant out more easily and then put all of the adjuvant into my tube. So now what I'm going to do is I'm going to take 2.5 mls of those and add in there on the vortex.
Since I took 2.5 mils of the adjuvant, I'm going to take 2.5 mils of my KLH. So as I add a drop by drop, you'll see the, the mix is going to change color and turn white. So now I've added all the, all the antigen to the adjuvant.
So what I'm going to do is going to vortex the mix for further five minutes. But since I don't want to hold the tube for five minutes, what I'm going to do is I'm going to tape it to the vortex To create the vortex inside the tube be you don't want it, you want it as fast as possible that it doesn't overflow. So I'm going to leave it like that for five minutes.
So the five minutes are over. I'm now going to stop the vortex and my mix is white. So I take the tube out.Oops.
The mix is completely white by now, but it's still pretty liquid. And I wanted harder for injection. So what I'm going to do is I'm going to transfer the entire mix into a five M glass syringe.
So those syringes have been autoclaved. So I link a complete syringe to my bridge. And from the other syringe, I only link the barrel.
I leave the plunger out and then I'm going to pour my mix in the top syringe. I'm going to transfer everything to the lower syringe. Then I need to chase the air bubble that I have here.
So I'm going to disconnect the top syringe and I'm going to chase the air bubble. Bubble is difficult to see, but it's right here. So I chase the air bubble.
I also chase it out of the bridge, and then I relink the top syringe. I make sure everything is tightly connected, and then I'm going to pass my mix from one syringe to the other. So at first it's easy, but soon the mix is going to become hard.
So tonight's becoming harder and I don't want to break the bridge, so I just turn my syringes. Sometimes it takes longer. It varies from emulsion to emulsion.
It probably depends on the temperature and level of, of humidity also. But I think my emulsion is ready. It's much thicker than it was when I poured it.
It's completely white. And there's only one phase. You should never have separation of the ACO phase from the oil.
So now it's ready to inject. So now the immersion is ready to inject. So to inject it, I use, I'm going to inject 200 microliters per hut.
And for that I'm going to use three mil syringes. And I use lu lock tip syringes to make sure that the needle doesn't pop out because the emulsion can be really thick and difficult to inject. And the needles I use, I try to use the smallest needles possible that we leave, let the emulsion go through.
And I found that the 20 3G one are good enough for this emulsion. For thicker emulsions, you might need to go with bigger syringes. Now the way we do it, we have one person hold the rat and the other person inject.
And for maximum safety, the person who holds the rats, although those rats are very calm and have been used to being handled, the person who holds the rats gets to use those rodent teeth proof gloves just for maximum safety we don't want to rat by. So I put all the emulsion in one glass syringe and I disconnect the empty glass syringe from the bridge. And in its place I put my lower lock three mil plastic syringe.
Now I transfer more than I need for one rut of emulsion. I chase the air bubble. Since I'm going to inject 200 microliters per rut, I live 300 microliters in my syringe.
I disconnect the bridge, I put my needle onto my plastic syringe and then I fill my needle and I put my plunger down to 200 microliters. That's way I'm sure I don't have air bubbles. And now we are ready to inject.
So we want to make sure the rat doesn't move. So we grab the rat and I put one finger above the shoulder, one below, and then with the other hand, I hold the back legs and the tail and we're going to inject in the flanks here and on microliters on this side and then microliters on the other side. So last week I immunized the rats with an emulsion containing KLH as an antigen.
And today I'm going to challenge them intradermally on the back with KLH, which is my antigen, and with o albumin, which is a relevant antigen, but to be able to inject them on the back intradermally first I have to shave them. And for that I'm just using a simple electric shaver. Yeah, well I change needles that in between each rats to make sure the needle is not blunt.
It makes it much easier to inject intradermally, especially I'm injecting small volumes of 20 microliters of each antigen. So I've injected my two antigens to all of the rats and I'm going to leave them in their cages until tomorrow. And tomorrow we're going to be able to see an inflammation.
So the site of injection will be red and swollen and we will be able to measure the diameter of the site of inflammation as a measure of inflammation. So yesterday I challenged the rats in Intradermally in the back with my relevant antigen, which is KLH, and an irrelevant antigen, which is of albumin. And now 24 hours later, and I'm going to look at the rats, look if I have an inflammation.
So I expect inflammation only on the KLH site and inflammation will show as redness and swelling. And after I've seen inflammation, I'm going to measure the inflammed area by simply measuring the diameter of the area. So this here is the KH injected site and you can see with no problem, there's a huge inflammation.
It's very red compared to normal skin and it's swollen. If we now look at the other site which receives of albumin, you can see the site of injection right here, but there is no significant redness or swelling. So the negative control worked.
So now I'm going to measure the site of inflammation. For that I'm going to use a regular ruler and I'm going to make a measurement in millimeters. This weighs six millimeters and I take a second measure.
This weighs five millimeters. There's very little redness, will take measurement, but it's barely one millimeter. So what I showed you here is how to prepare an emulsion with an antigen, how to immunize rats with this emulsion, and then challenge them week later with the antigen I used for immunization and also in relevant antigen and how to visualize and measure the degree of inflammation in used and in the Chan lab.
We use this technique pretty routinely to as a simple assay to measure the effect of potassium channel blockers on inflammation in rats.
