Voorbereiden van T-cel groeifactor van Rat Splenocyten

Hello, I'm Christine Beaton. I'm an assistant researcher in George Chen's lab in the Department of Physiology and biophysics of the University of California at Irvine. And in the next two days I'm going to show you how to prepare TCGF, which stands for T cell growth factor.

So for our everyday cell culture, when we do not need a precise knowledge of the composition of the cytokines in the culture medium, what we use it's T-cell growth factor, which is S supernatant from spl CY culture that contains the required cytokines to help the T cells expand. So the way we prepare the TCGF is when we kill healthy Louis rats, we take their spleens, prepare single cell suspension, get rid of the red blood cells by ly them, and then the mono nuclear cells we're going to stimulate with con carnaval in a, which is going to induce the T cells to produce all the required cytokines for their growth. To generate the TCGF, we need to activate the cells.

So the way we do it is we're going to add lectin conval in a abbreviated as corona A to our cell culture. So when we add the conc in a to our cell culture, it is going to bind to the glycosylated T-cell receptor complex and binding to several complexes at the same time, it's going to do what we call capping. So it's going to aggregate, make a cluster of all the receptors at the surface of the cell and that's going to induce an activation signal in the T lymphocytes.

So after the cells have become activated, they're going to blast. So they're going to enlarge and then they're going to start secreting cytokines. So originally we thought that TCGF was mainly interleukin two, but we now know that it's not really the case.

It also contains interferon gamma TNF alpha and probably also cytokines in very small amounts that are just necessary to keep cells alive after 48 hours In culture, the cells have produced all the cytokines we need. So we are going to remove the cells and when we have remove the cells and we just have the supernatant, we still have some free conal in a in the snat. And that is going to be a problem because we really want to use our T-cell growth factor to keep T-cell lines growing.

And if we leave the conval in a free in there, it's going to activate the cells when we don't want them to get activated. So to get around that problem, we're going to add an excess of a sugar that's going to bind to all the free conc in a, in the ate. So it'll be completely inactivated and will not bind to other glycosylated receptors on the cells when we add the TCGF to the culture.

So let's get started. The first step for this experiment Is to take out spleens from rat right after euthanasia. So that's what I'm going to do right away using sterile instruments.

And once I have the spleens out, I'm going to put them in PBS supplemented with antibiotics and as antibiotics I use penicillin and streptomycin. So let's get started. So I spray down the rat with 70%ethanol to ensure I don't have a tall any contamination of my culture.

And then using my instruments, I'm going to make a small cut in the skin here. Then I remove layer of muscle and the spleen appears right here. So I take other tweezers, I gently pull out the spleen without damaging it.

I remove the connect connective tissue like this. So now I have a spleen. Of course, I'm very careful not to damage anything else inside the abdominal cavity because any damage to the intestine is going to lead to immediate contamination of my spleen.

And then I'm going to transfer my spleen into a tube containing PBS plus antibiotics. And this tube I keep on ice. So now I have harvested the spleens from three rats.

So we are ready to go and prepare a single cell suspension from those spleens. So now that I have my Spleens, I brought them to a tissue culturehood to prepare a single cell suspension from them using 70 micron cell trainers. So I'm going to need several things to make the single cell suspension from the spleens.

I need sterile instruments, one pair of forceps and one pair of scissors. Then I have the plunger of a sterile one mil syringe in the Petri dish. I'm going to put my spleens in PBS plus antibiotics.

And in another Petri dish I've put 70 micron cell strainer in 10 milliliters of PBS plus antibiotics. So the first step is to make sure that the spleens are clean because as you can see here, there are still little pieces of conjunctive tissue and I'm going to remove them because they're going to clog cell strainer really fast. So for that, I simply cut off what I don't want and discard it into the lid of the Petri dish.

So now my spleens are mostly clean, so I'm going to take the spleens one by one, put them in the cell trainer and cut them in small pieces And I mix all three ples. The next step is to make a single cell suspension. So of course since I want everything ster, I don't touch the cell trainer at all and I use the plunger of a sterile one mil syringe to press my pieces of spleen through the cell trainer.

And as you can see on the outside of the cell trainer, I have a single cell suspension being made. So the first time around I still have some small pieces of spleen, but I'm already going to take out the single cell suspension. I already have transfer it into a 50 mil tube that I have stored on ice.

And I'm going to refill my cell strainer with 10 more mils of PBS and antibiotics. So I take 10 more mils of my PBS with antibiotics approximately, doesn't have to be very precisely 10 minutes, we are just washing. I put it in the cell strainer and then I use the same procedure.

I go back to my syringe plunger and I press through the ocid a little more. So of course you'll never get everything through because there's conjunctive tissue and small pieces of fat I wasn't able to remove that are going to remain in the cell strainer. But you should get the majority of your spleen for the cell strainer.

And then I repeat exactly what I did earlier. I'm going to harvest my single cell suspension and add it to the same tooth. By now there's almost nothing from the screen left in the cell screener and the PBS that comes out is much clearer And I am going to simply wash once more and we should have most of the cells out by now.

So now I have my single cell suspension of PLE cytes and I'm going to spin them down simply to palette them. So spinning for eight to 10 minutes will be enough. So now we've spun down our PLE cytes and we have a nice palette.

So what I'm going to do is get rid of the supernatant and I'm then going to ly the red blood cells. So I'm going to suspend myocytes and I'm going in ammonium chloride. So it's a SU a solution of oh point 15 molar ammonium chloride and I'm going to use five milliliters per spleen.

So since I had three spleens, I'm going to use 15 milliliters of ammonium chloride and I'm going to do that on ice. So to lies the erythrocytes, I'm going to gently pipe it up and down for three minutes. Okay, we'll use this take with the higher exposure here.

So now I've analyzed the electrocytes for three minutes. I'm going to fill up my tube with PBS or you could use medium also. That's to bring the osmolarity of the solution back to normal range for the cells.

And I'm going to spin the cells down like I did previously for eight to 10 minutes to pellet them. And then I'll wash them twice using the same PBS with antibiotics. So now we span down the cells after izing the electrocytes and don't be worried we never get a hundred percent releases of the electrocytes.

So the pallet will still be red, but the supernatant will be clearer. And now I'm going to empty the supernatant, break the pallet, add in some PBS up to 50 mil, spin down and repeat that step once more so that I'll have two washes And then I'll be able to count myself. I have washed myocytes Twice and then I've suspended them in culture medium and I've counted them using your cytometer.

As expected. From three spleens I got around 600 million cells, which is expected since one spleen normally gives us 200 to 250 million cells. So what I'm going to do now is I'm going to seed my cells at 2 million per mill in my medium that has been supplemented with 10%fetal cal serum.

And to that medium I'm going to add two microgram per mill of conc A to stimulate the cells. And after that I'm simply going to put the cells in the incubator for 48 hours. The cells have now been in the incubator for 48 hours and as I'll show you, they have grown pretty well.

The medium has turned orange. So we are now ready to finish our preparation for the TCGF. As you remember, I added con carnaval in a in my cell suspension to activate the cells.

So the con carnaval in a is lectin. It's going to bind the sugars on all the receptors of the of the T-cell and it's going to artificially activate all those cells. The problem we have now is that we still have some of the con carnaval in a, in the cell S supernatant.

And since we're going to use a snat in other T cell cultures to expand the cells, we do not want the con carnaval in a present in those cell cultures. So to get rid of it, what I'm going to do is I'm going to add an excess of sugar to which con naval in a binds, and that's methyl alpha D cyte shown here. And I'm going to add an excess of this sugar completely dissolve it.

It's going to bind all of the remaining conc naval in a And once that's done, I'm going to sterile filter my cell supernatants and I'm going to freeze Ali liquids for use in the next few weeks or months. So let's get started for this last step. So I'm adding my cell culture supernatant to 50 mil tubes to be able to spin them to get rid of the cells.

So I simply fill as many tubes as necessary and I don't do that in the culture hood, I could, but since I need any way to filter my, my solution in the end because I'm adding the sugar, which is not sterile, I simply do that on the bench. It's easier. I've now poured all my cells and supernatant in my 50 mil falcon tubes.

So I'm going to bring them to the centrifuge and spin them for about 10 minutes. And the only reason for spinning them is to pellet the cells so that I'll have clear supernatants. The cells have now been spanned down, so the supernatant is clear and that's what I wanted to collect.

So I'm going to collect all the supernatant into a clean 150 square centimeter flask. And to this flask I'm going to add the sugar. So while the cells were spinning down, I weighed enough sugar to add 15 milligram per ml of supernatant.

I'm going to completely let, let it dissolve, and after that I'm going to sterile filter my supernatant. So now I'm going to add the sugar 15 milligram per ml of supernatant. I put the cap back and since I'm not working in sterile conditions, it doesn't matter if ate goes into the the cap, so I mix until all of the sugar is dissolved.

That should be pretty fast. Yeah, all of the sugar is dissolved now. So all I have to do is sterile filter is supernatant and then I'm going to aliquot it.

So normally I do 10 to 15 mil aliquots that I store at minus 20 degrees. Aliquots like that can be stored for several months. You can also store your TCGF in the fridge, but then I would use it within the next 10 to 15 days.

Over the last two days, I have showed you how to prepare T-cell growth factor, and this supernatant is very simple to make and it's very cheap to prepare. So we use it very often in the lab to maintain our T cell lines in culture without having to buy expensive cocktails of cytokines. So good luck With your experiments.