人类神经干细胞转染与Amaxa Nucleofector

Hi, I’m Steve. I work in Dr.Flanagan’s lab at the Department of Pathology at uc, Irvine. Today I am going to show you how to transfect human neural precursor cells using a maxis nuclear factor.

First to start, you’re going to need a lot of cells. I would say at least a confluent T 25 flask as this protocol requires a very high cell density, so you’ll need at least a million cells. First, I’m going to start by taking the cells off the surface using cell dissociation buffer, and then counting them.

And for that procedure refer to my packaging protocol. And the reason why I prefer a max maxa for my transfect experiments is because amaxa develops tailormade transfection protocols for specific cell types, and in my case, for neural stem cells. And it works great.

It gives very high transfect efficiency and very high cell viability. And let’s get started. So now I’m ready to do a transfection.

So in preparation for this transfection, I’ve already coded my tissue culture dish with fibrin overnight. And after that, I placed some growth media in it. So right now it’s sitting in the incubator waiting to receive the cells.

Also, I’ve prepared all, all my reagents, my DNA vector and the transfection solution. So they’re all waiting here on ice. And I’m ready to begin the transfection.

So this is the M max nuclear factor, and before I place it into the hood, I’m going to disinfect it with 70%ethanol, and then I’m going to wipe it down with a paper towel. So I’ve just counted myself so that I have 4 million cells total and spawn them. And now I’m ready to start transecting.

So first I’m going to remove the media that they’re in. So I have my cell pallet right here, and I’m going to suction off the media that they were in. And I am going to try to remove as much media as possible because I don’t want to have any proteins or such in the transfection media because that might affect the transfection protocol.

So I’ve removed pretty much most of the media, and now I’m going to resuspend this cell pallet in a hundred microliters of this specifically formulated mouse neuro stem cell nuclear factor solution. Mxa makes a transfection solution for transecting mouse neuro stem cells, and they don’t have one for human neuro stem cells, but we found that using the one for mouse cells works very well. So I’m going to qu a hundred microliters of this mouse neuro stem cell nuclear effector solution.

And I’m going to reus suspend my cell cell pallet in it very well, so that there are no clumps of cells remaining. So that pretty much, I have a homogeneous cell suspension. So I’m going to pipe up and down about 10 times.

So I think now I have a pretty homogeneous mixture, and I’m going to transfer it now into a 1.5 mil open dorf tube. So I’m using a P 1000 pipette and am going to make sure I get all of this solution. So now I have my 4 million cells reus suspended in mouse, new mouse transfection solution, and now I’m going to add five microliters of my vector.

So I’ve been keeping it on ice all this time until now. And so I’m going to, first I’ll quad five micro microliters. So now I’m gonna add it to the cell suspension.

Now I’m going to use a P 200 set to about 50 microliters, and I’m going to mix the cells with the vector really, really well. So I’m going to pipe it up and down 10 times. Okay, so that’s ready.

And I, I am ready to transfect. So I’m going to open a new ette. And also I’m going to get a, a special pipette provided by amaxa.

So I’m gonna remove the cap and I’m going to gently pull up this mix. And I am going to insert the pipette all the way into the vete and gently squeeze out the solution. So I’m going to turn on the max nuclear factor, and I’m going to set this to a 33 0 3 3 going to press enter.

Then I’m going to place the vete into the vete holder, and then I’m going to rotate the wheel, and now I’m ready to transfect. But before that, I’m going to make sure I have warm media ready to be added to the transfected cells right away. That helps increase cell viability if I add warm media into the, into the vete right after a transfection.

So I’m going to prepare a P 1000. And so, and also I’m going to get the played with media that’s sitting in the incubator right now so that I can transfer the cells into the media right away. So now I’m, now I’m going to press enter again and it’s done.

And the successful transfection, as you can see, you have here this very thick layer of bubbles that indicate successful transfection. So I’m going to add 500 microliters of prewarm media into the vete. So I’m adding more media that the cells desperately want, and I’m going to use the same MX pipette to get the cells out of the vete.

So they’re really stressed out right now. So I’m going to put em directly into warm media, more warm media. And finally, the last step, I’m going to rinse the Q vete with some more prewarm media to make sure I get, I get all of these cells out, and I’m going to place it in this into the same well L and place the plate into the incubator right away.

In this image, we see human neural precursor cells that have been transfected with a access nuclear factor and plated on grided cover slips. So even two days post transfection, we have about 50%transfection efficiency that we can observe by GFP expression. Today I showed you how to transfect human neural precursor cells using Xis nuclear factor.

Good luck with your transfection.