Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

So these are the first type of beads I'll use. They are the gel blue gel beads. They're soft.

So I need to use a pooled glass pipette at the end of a mouth pipette to place these beads on Xs.The second type of bead that I'll demonstrate are heparin acrylic beads. They are a stiff type of bead, and I will place these beads on X explants using A tungsten needle. So these are the Glass capillary tubes that I'll first melt using the bunsen burner.

Then I'll pull them to create a finer tip. So I hold the capillary tube in the middle of the flame and I roll it as the tube melts. I can feel it get soft.

I'll lift it out of the flame and then pull it. I am going to etch a line across the capillary tube with this diamond tipped pen so that I can have a nice straight cut. Then I will flame polish the ends to clean it up.

I'm going to pass the capillary through the flame to polish the end and make the opening smaller so that it can capture a bead rather than the bead passing through the capillary. An important thing to note is that you don't want to hold the capillary tube in the flame very long. It's actually a very quick pass, otherwise it'll seal.

I usually make several of these because many of them turn out where the tip is sealed by the flame. It's hard to control, so you just make several. This is an example of one where the tip has been sealed.

This is an example of a good pipette. I have pipetted 250 microliters of the agel beads into this einor tube, and I'm going to wash them five times with one milliliter of PBS to remove the azi solution that they were soaking in. I'll wait about three minutes to let the beads settle and then I'll aspirate off the excess PBS and repeat this four more times.

The beads haven't quite settled yet, so the beads have settled and I'm going to aspirate off the excess PBS and repeat this four times. And at the end I'll add 300 microliters of the PBS and begin to work with the beads. This is a standard mouth pipette and I'm just gonna take the capillary that I pulled and push it in there about a third of the way up.

First, fill the pipettes with PBS. It usually fills just by a capillary action. So this is a lid off of 30 millimeter culture dish and I've put a droplet of beads in blue.

And next to it is a small droplet of PBS. And in this dish here is a five microliter droplets of protein solution. In this case, it's B and P four, but you could use a protein of choice using the mouth pipette.

I'm going to transfer a bead from this blue circle here, rinse it once in the droplet of PBS. Then I will take the bead with the mouth pipette and place it into the droplet of protein solution and incubate it either room temperature for at about an hour or four degrees for several hours. So there you see a pool of beads in PBS and I need to select beads that are the right size.

If they're too small, they'll simply go through the mouth pipette. They need to just sit at the end. So now you see that I've chosen a few beads there, three larger beads and three smaller beads.

I'll use the larger beads in the experiment. I'm gonna now transfer a large bead from the droplet of PBS over to the droplet of protein solution. And to do that, I'm going to gently pull my tongue back away from the mouth pipette.

It's not a suck sucking motion, but just to apply some negative pressure. And then I'm gonna transfer it over and then basically place my tongue back over the mouth. Pipe head, not blowing, but just adding some positive pressure to to push the beat off the end.

I now have five beads in the protein solution droplet, and I'll incubate them at four degrees for several hours, or alternatively at room temperature for a half hour to an Hour. So this is the culture Dish that contains the membrane that's floating in media along with the eggplants that I've already dissected. I'm going to take the tip of my pipette and suck in some of the media.

I am going to rinse the beads in a droplet of media just one time immediately before I place the bead onto the X explan. These are the tungsten needles That I will use to pick up and place the acrylic beads. This is the tungsten needle and it's in its holder now.

And these are some fine forceps. At a 45 degree Angle. I'm gonna use my Tungsten needle in forceps to lift a bead from this pool of PBS and transfer the bead over to a droplet of protein solution.

As I did with the agel beads. Like with the agel beads, I've rinsed these acrylic beads five times in PBS and transferred a droplet to this plate. I'm going to then use my tungsten needle in forceps to transfer a bead to a droplet of protein and incubate just as I did with the gel beads.

So it's a lift the bead. I have my right hand firmly against the the table, and I use my left index finger to steady the the needle as I go down into the fluid, I lightly touch a bead with a needle and lift it slowly out of the fluid. Sometimes it drops off the needle, so just go back for another one.

Now with my left hand, I have the 45 degree angle forceps. With the points down, I touch the bead and the needle to the fluid and use. Use the tip of the forceps to gently just press it off.

So now I have Five beads in protein. Okay, So with my right hand I have the tungsten needle in the bead, and my left hand, my forceps again with the point down the same way that I push them off The four, I'll do it again here on the x explan. Incubate at 37 degrees and the time depends on particular assay.