血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

Hi, my name is Mar Katsu. I'm from the Hughes Lab in the Department of Molecular Biology and biochemistry at uc Irvine. I'll be performing the fibrin bead assay.

The entire process takes two days to complete. The first day involves coating beads with pubic or human umbilical vein endothelial cells. The second day involves embedding these coated beads into fibrin gels and then layering fibroblasts on top of these gels.

We use this assay to study the many steps Of angiogenesis in in vitro setting. First day in part one we use CY X three Beads. And then the second day we use these three reagents, fibrinogen thrombin, and aprotinin cytx.

Beads three are from Amazon Biosciences and they're especially collagen coded so that the endothelial cells stick to the beads. So the next reagent is fibrinogen, and we get this from sigma. This forms the actual gel or clot in the beat assay.

Thrombin is a coagulation factor. This protein converts fibrinogen into fibrin, so that's why we add it into the assay. Aprotinin is a serum protease inhibitor.

We use this to prevent Proteases from degrading the fibrin gel. So what you see here are vac. These are the Cells that we use for the beat assay.

This particular cells, these cells are at patches three. We normally use them from patches two to patches three. You can see that these heve take on this distinct cobblestone morphology.

Right at this point I believe these cells are at a hundred percent co fluency and you'll want to use these cells at this Fluency. So this is day one of our beat two assay and this Is how we do it. Basically, just wanna flip the tube several times just to get the beats and suspension like so, and you'll do this every 20 minutes for four hours.

And that's pretty much it. Okay, so this is the second day of our A b assay and these beads have just been coated with endothelial cells. What you see here is basically a highlights the difference between a non coated bead versus a coated bead from one.

The left is obviously a non coated bead. You can tell by the bumps on the surface of the bead. The coated bead should look like mini golf balls.

You can see that it's the distribution. The bees is pretty uniform. You really shouldn't see any non-coating bees.

You might, you might see one or two like that one right there, but for the most part, majority, I'd say 99%of them should be coated. So these are the bees that consider in an incubator Overnight. What I'm gonna do is I'm gonna transfer the media, which contains some deeds to a 50 mil conical tube.

I'll then wash the FLA to remove the excess feeds that stuck to the bottom of the PLA and transfer em to the conical tube as well. Beads tend to stick to the to the flaz. So what I'm gonna do is I'm gonna take another five mils of EGM two or media and wash off these stuck beads.

So now I'm going to transfer these wash beads to the conal tube. So after you transferred your media with beads to the conal tube, the beads settled to the bottom of the tube in about a couple minutes. So it's hard to see, but there is a small layer of beads at the bottom of this too.

So now I'm gonna aspirate off the old media. Okay, so now I'm going to wash the beads with a mill of e GM two and then transfer it to an fendor tube. So what I'm doing right now is I'm washing the beads and you wanna be very, very careful because the beads are very fragile.

So you want to pep it at a very slow speed. So just do it like this. The layer of beads is really hard to see though.

Just give it a couple minutes and all the beads, you settle to the bottom. So now I'm gonna do is I'm going to aspirate off the old media and wash it with another mill of EGM two. And you're gonna do this three times.

You don't want to aspirate all of the media about to this level is good. So again, you wanna pipe it up and down very slowly. So this process is You have to work quickly because what I'm gonna do is I'm going to take an L quad to mic, 10 mic liters of our bead solution and transfer it to cover of, to count the number Bs to get a concentration of beads per mil.

The beads settle quickly to the bottom of the two. So you have to work fast. I'm gonna for it off gold media.

And again, you'll have to work very quickly. I also have my pipettes all ready to go too, just to everything streamlined. Now I'm gonna wash the beads again, pipe up and down really slow.Okay?

And then right after that, take your other pipe, B 20, add two 10 microliter. Quas on a cover slip, like so we take two because I like to get an average of of bulk drops. We've just transferred two quas of 10 microliters of our feed solution to this cover slip.

And this is a process in how we quantitate the number of beads in our one mil of EGM two. Basically, you just count the number of beads that you see, and I usually do two, take two elk quads and calculate the average from the tube to get the concentration of beads per microliter. So I've counted a number of beads in our tube and I have about around a thousand beads.

I've already made up my friedgen solution. It contains also a protein in and a final concentration of 0.15 units per mil. And I have 2.5 mils of friedgen.

So this will give us a total of five wells since each well volume is 500 microliters. So to get the beads into the fibrinogen solution, I'll first ask rate media. I'll take an qu about five, 600 microliters of our fibrinogen and use this to mix and transfer the beats to the fibrinogen tube.

So now what you wanna do is you wanna take about 500 600 microliters of fibrinogen and use this to mix the beads and transfer them back to the Tube. So now before You add the FibroGen to the, well, you want to add thrombin first. Add a concentration of 0.625 units per mil, and you're gonna add it directly into the center of each well.

So now you see I've added thrombin to each well, and they're directly in the center. And now the next step will be adding the fiber engine. So the bees, again, remember they settled to the bottom of the tube rather quickly.

So I'm gonna take my fiber ular L quad, but before that I'm gonna mix the beasts like so, just to get em into solutions so that they come off from the bottom. You know when you want to pipe it up and down slowly. So this is the most important step in the protocol.

First of all, you wanna make sure that you pipe it up and down slowly while you're adding the fiber into the center and the thrombin. So place your pipette tip direct in the center and pipette up and down while you're doing this. You don't wanna move the plate at all cause any s slight movement can actually create effect where the, the beads actually clump into the center of the well.

You want it evenly distributed. Pipe it up and down several times like so, very slowly so you don't create bubbles. Bubbles can actually hinder the visibility of your gel.

So you wanna be as slow as possible and do not move the plate and switch pipette tips every time you add fiber to the wells. We're gonna let these clots solidify for five minutes. We'll then transfer this display after five minutes to the 37 degree incubator, another 10 to 15 minutes.

It's been five minutes. The fiber has formed the clot. And what I usually do is I just take a quick glimpse to see if the beads are even distributed and going from well to, well, you look pretty good.

There's about, I'd say 200 beads per well, on average you want to have it between 200 and 250. What I'm now gonna do is I'm gonna stick this into the 37 degree incubator and incubate It for about 10 to 15 minutes. So these are the long far blasts That we're using to co-culture with our IC in the beat assay.

This is a confluent flask. You notice that the blass are very different morphology compared to the beck. They're more skinnier and they look almost like strings.

What we do is we just basically ize this flask, harvest the lung fire blasts and layer 20, 000 fire blasts per well. I've just ized the fire blasts and I've suspended em at a concentration of million cells per mil or a thousand cells per microliter. Now I'm gonna add it to the plate.

I've just taken out our plate from the incubator. It's been 10 to 15 minutes and the fibrin is formed a complete clot. But what now I'm gonna do is I'm gonna add a mill of EGM two to each well, and I'm gonna do this dropwise because if you do add the e GM two to the each, well too fast, you could actually tear the gel.

So this basically do a nice slow pace move from one well to the other. So I've just trinia to fire blasts and I've counted them and I suspended the, the fire blasts usually at a concentration on million cells per mil or a thousand cells per microliter. And what I'm gonna do is I'm gonna take 20 microliters and add it to each well.

And while you're doing this, you want to switch pipette tips every time. Take another qua of the fire blasts and just add the fire blasts directly into the center of the well. And again, I'm changing the tip every time I add fire blasts.

So I've just added the fire blasts on top of the fibrin gel. And the fiber blasts are in a mill of e GM two. The gel contains the coated beads and you can actually see a couple beads as we focus into the plane.

After a couple days, the fiber blasts will eventually proliferate and and migrate forming a monolayer over the fibrin gel. A couple days after that, around around day three, day four, you start seeing Some nice sprouting coming off the beads. So I just finished performing the fibrin bead Assay and to recap, what we did is we coded endo saal cells on beads and embedded these beads and into fibrin gels and then layered on top of these gels.

So over the next three or four days, we just started seeing some nice sprouts coming off these beads. And what I really like about these video protocols is that viewers can actually visualize these steps that you really can't convey through written protocol. So what I'm hoping what the viewers see in these, in these videos is that they'll be able to perform these fi BTA SC successfully in their own lab.