Hello, my name is Sochi Agata. I am a postdoctoral scientist in Dr.Ken Cho's lab at development of developmental and cell biology in University of California Irvine. Today I will show the dissection and adhesion assay of animal cap explan or organizer expand from frog XUS embryo.
This procedure can be, can be utilized to study the cell adhesion characteristics and cell morphology of the cell derived from dissected tissue. This procedure involves in the dissection of a certain tissue from the xenopus frog embryo and they dissociated in calcium magnesium free medium and then bring that dissociated cell to the calcium magnesium containing containing condition on top of the cell adhesion, molecule coated slide glass. And you we can study the cell behavior, cell migration and adhesion characteristics of the dissected X one in this procedure.
Okay, this whole procedure of dissection of embryo and adhesion assay, I use foreign special tools first. This is a calcium magnesium free one x-bar, 1%agro sprayed. And this is a just right here, one x-bar, 1%agro sprayed on which you dissect the embryo.
And then this is a dissection, special dissection tool other than forceps. This is called eyebrow knife, which is weird name, but this is actually literally made of your eyebrow pulling off from your face. And then stick on the tip of pasta pipette.
And this the other tool here is called hair loop, which is again your hair making a loop on the tip of past pipette. I didn't use this tool this particular time, but some people prefer this hair loop more than eyeball knife and therefore adhesion assay. I use the silicone rubber insulator on which stick on top of the slide glass to make adhesion react adhesion assay chamber.
So first take vine envelope out from the embryo. This piece is vine envelope. Then dissects the animal pole ader.
This is animal proect, you don't need it. Flip the embryo over and you'll see that clear dorsal lip here. It's beautiful.
Then take eyeball knife, stick to the side of dorsal lip, stick the other side too and then cut this piece just below the dorsal lip. And this piece contain organize the me and surrounding derm. You flip this and you peel the abdominal part of out from internal organizer.
Me.Okay, I kind of destroy the dermal part but that's not important. So that's okay. And this piece on the right hand side is the organizer measure.
I just moved to the cleaner side of the dish. The piece on the right hand side is organizer mera piece and this is the whole embryo. And now you see the how, how, how, how much is the size of the organizer piece look like.
And this can be used as a extracting some factor expressing the organizer region or make organizer CD in the library or study the thermal ology in the migrating romal tissue. Before dissecting animal cap ome you have to find the right stage. We use stage eight blast embryo.
And now here we see in the view is three the embryo at three different stages. The two embryo at the left hand side is stage seven blast and the M two embryo in the middle is stage eight. And the right hand side two embryo are stage almost stage nine late blaster.
And we like to see, we like to use stage eight the middle too because you see, first of all, you see the clear difference of each blast mare at the animal pole. And this stage eight embryo gives you the good combination of nice cell sites and easy easiness to dissect at stage stage seven. The animal pole we cell are too large and the number are too small, whereas the stage nine embryo each cell are too small and also the animal pro exome tissue are become thinner than stage eight.
So that you're not gonna get as many as many cells as you want. First you stick the forcep to the side of the embryo and take the vine envelope out from the embryo. This is vine envelope.
And then you dissect the center of animal pole out from the embryo. Okay, this is a beautiful piece. Left hand side is the dissected animal cap explan and this is the rest of the embryo.
And this piece can be, the usage of this piece is, for example, if you inject something to the animal pole of embryo at the earlier stage like the Porto gene construct, then you dissect this piece out in the, at the later stage and use it for the Porto assay like reciprocate assay For example. Now I am going to use transplant and transfer the organizer x explan to study the cell adhesion assay. And to do that I need to dissociate dissociate the organizer X explan in calcium mechanism three medium.
So I'm transferring from transferring the organizer explan from this one regular birth dish to calcium magnesium free medium dish. But to do that, if you're very good at your hand, you can use this regular transfer pipette but you have some remote chance of damaging embryo while doing so. So I, what I usually do is use P 200 and yellow tip.
And this yellow tip is previously coded with this chemical code single code to prevent the cell stick to the side of the yellow tip. And also I cut the tip with scissors at the tip of the yellow tip so that the tip is now dull and large bore so that I minimize the chance of damaging DX explan. Okay, now what you see in the view is the two organizer explan and I'm, I am going to transfer these two pieces from this one one express regular dish to calcium mechanism free medium dish.
Slowly suck those pieces and transfer to the calcium magnesium free medium. And now how, that's how they look like at the beginning and we now are now they dissociate almost the single cell level. Now the organized explan are in calcium magnesium free medium and I will incubate them in calcium mechanism free medium for an hour to allow them to dissociate to the single cell level for the next step to study the cell cell morphology or behavior after an hour of incubation the cell, I mean the organized explan supposed to be dissociated.
They are pretty well. And now take a look at the micros and the microscope. They are, yes, they look pretty Action.
Okay, now Im am studying the cell behavior on the slide imaging chamber which caught it with cadherin or INE or other type of cell adhesion molecule to make a cell adhesion imaging chamber. I take this slide and also this silicone rubber insulator and simply just put the silicone rubber insulator on the surface of slide and firmly press to make sure they stick all the way around of this circle. And then look from the other side to make sure that it stick all the way so that the liquid is not gonna leak on top.
On on on this sand chamber I pour the 200 microliter droplet of cell dehe, the suspension which contains cell adhesion protein like cohering or fibronectin. And after that block this chamber with 500 microliters of 4%BSA. And then we replace A BSA with regular one x bus, a fian medium.
And now it's look like here's a chamber ready to transport the cell. So this chamber, I take this again, this a P 200 attached with chopped off yellow tip coated with sigma code and suck up the cell from calcium magnesium free medium to this chamber, which contains the medium, which also contains the calcium and magnesium. Now, okay, now I'm transferring this dissociated organizer X point using P 200.
First you stick this P 200 and slowly suck the cell and you can pipe it up and down very gently. Still, I'm kind of spreading the cell everywhere, but to make sure that they are single cell, then bring this imaging chamber on the center of your view, then spread the cell everywhere in the imaging chamber. Now cell are spread on the coated coated surface.
And let's see, in the higher pipe the cells look like this. Okay, after two three hours spreading the cell onto the adhesive chamber, I count the cell on this chamber using this microscope. Typically when you try to Study the adhesion of organ cell from organizer explant, you leave the cell on top of coated slide for two to three hours.
And then after that you can quantify the number of the cell on the slide under the microscope to wash. Wash out the DO three attached or untouched cell, you just simply put this coat on the hession chamber into the buffer bucket, which contains large quantity of buffer. And then start the agitation at very mild speed, like 50 RPM, which is about the speed, And wait for five minutes.
After five minutes, you Carefully take this adhesion chamber out without exposing the cell inside of this adhesion chamber. So you carefully take this out like this, then wipe this off excess buffer and study under the microscope again to count how many percent of the cells stay attached to the coated surface. Now after washing, put this adhesion chamber back to the same spot to make sure that I am observing the same exact location of the adhesion chamber.
And now Start counting. So on this screen you show I I I'm showing two Different type of cell on the third same type of cell adhesion molecule. And those two types of cell shows completely different adhesion characteristics on the same pro.
On the on top of the same adhesive cell adhesive protein. We could also use this dis associated cell from the explan for their aggregation or migration behavior in Time lape imaging.
