This is a Max MIDI magnet. It says, caution extremely strong magnetic fields. Hi, I'm Melanie Matthew from Mike Collins lab at the University of California Irvine Department of Physiology and Biophysics.
Today we're gonna show you a negative selection preparation of CD four positive T cells using the Milton biotechnology kits. So here we have the minimax and the minimax magnets and the minimax are used for larger separations. You can run up to 10 to the ninth cells through a column that fits in the minimax and the minimax are used for much smaller volumes of cells.
So depending on whether or not you're doing a positive or a negative separation, you will label your cells with the appropriate antibody. They have a magnetic beat on them that will attach to the magnet. In the case of a negative selection, the cells that you're interested in will run through the column and you can collect the EL pollutant, which will contain your cells of interest.
I should mention the techniques I'm showing you tonight are not sterile. And so if you wanna use these cells for cell culture, you should do everything in the tissue culture hood with sterile instruments. So even though I'm showing you a preparation for CD four positive T cells, many of these techniques are applicable in purification of other cell types using the milit tiny biotechnology kits.
Okay, so I've removed lymph nodes from the mouse, and I show you how to do this in detail in issue seven of Joe lymph node preparation for two photon imaging. And now I'm going to grind the lymph nodes up into a single cell suspension inside of a 70 micron cell strainer. Okay, so now I'm going to grind the lymph nodes into a single cell suspension using the back of a six milliliter syringe.
The lymph nodes are in RPMI inside of a filter, and it's important to make a proper single cell suspension. And the reason we're doing this in a filter is that you wanna make sure you don't have any cell clumps. So when I'm done grinding, I'm gonna take the filter outta the media and then use the alls that have come through the strainer and the single cell suspension.
When you're grinding, you wanna make sure that you disrupt all of the tissue as much as possible. This single cell suspension is what we will purify our CD four positive T cells from. Okay, so now I'm done grinding the tissue and you can see that most of the lymph nodes are pretty much gone.
We still have some small bits of fat and kind of the shell of a lymph node here with a little bit of connective tissue that's all stuck together from all six lymph nodes. And now I remove the cell strainer. And you can see here that the media is very cloudy.
It's full of cells from the single cell suspension to ensure that we get a maximum number of cells from the filter and some of them will stick to it. I wash it with a couple mils of media and then I pipet all of the media into the 15 mil Falcon tube and I spin this down to pelt the cells through later purification. So here's my single cell suspension, spun down and pelleted at 300 G for 10 minutes.
If you think you're going to have a high population of dead cells in your single cell suspension, it's a good idea to use the dead cell removal kit from Milit Tene. So now that I have this pelted down, I'm going to pour off the media and do a red blood cell lysis, and then I'm going to count the cells and then do a CD four positive T-cell negative selection kit on these particular cells. Okay, so now I'm going to do an RBC lysis.
We're going to lyce the red blood cells that we've pelleted along with the lymphocytes by decreasing the osmolarity of the solution by first adding water and then bringing it back up to a one x solution by adding 10 X-D-B-P-S. Our lymphocytes are more resistant to the change in osmolarity than red blood cells, but we still have to work quickly or will ly our lymphocytes, which we don't want to do. Part of working quickly is that I fill the volumes I'm going to need for the lysis prior to actually adding water to the system.
So I wanna be able to add my 10 X-D-B-P-S-A few seconds after I add my water to make sure that I don't lyse my lymphocytes and I adjust lys the red blood cells. So I take 900 microliters of water. It's almost a mill and a hundred microliters of 10 X-D-B-P-S.
I have both pipe ready to go. In the lysis, I add the water, wait a couple seconds, maybe shake the tube a bit and then add the 10 X-D-B-P-S, shake the tube a little more. And if you have a lot of red blood cells that you're lysine, you'll see a large kind of amorphous blob form of all the broken cell membranes.
We have a little bit of it here and you can see that it's a little bit of broken cell membranes. So after I've done the lysis, in case there's been any mistake pipetting, I like to add a few mills of media to the cells to make sure that the osmolarity is correct and we don't continue anymore of the lysine. Okay, so now I have my lymphocytes in the single cell suspension.
Again, I've done the red blood cell lysis and I'm going to spin them down and pellet them. Before I do this, I'm going to count them on the hemo cytometer to determine how much antibody we need to add for the CD four positive T cell negative selection kit. Okay, so now I spend down the cells in max buffer and you can see that the pellet is a lot more white After we've done the RBC lysis.
And I'm going to do a negative selection for CD four positive T cells. So I'm gonna pour off all the media, all the max buffer that I spend the cells down in. And I'm going to label with the biotin antibody cocktail, which is step one in the negative selection for CD four positive T cells.
And then I incubate in the refrigerator for 10 minutes. Okay, so my cells are done with their initial incubation. Now I'm going to add the antibiotic to microbeads and put them back in the refrigerator for 15 minutes.
So for this particular cell separation, we use an LS column from Milit tiny biotech and we, what we're going to do is put it on the magnetic holder and prepare the column by running three mills of max buffer Through it, we're going to open the column and place it so that the prongs face inward into the magnet. Now I'm going to add the max buffer to the column, and you should see it go down the LS column and start dripping out at the bottom. Don't worry about the column running dry.
The surface tension of the liquid will keep it from running dry until you add your cells. Before you add your cells, it's important to let the max buffer drink completely through the column. Okay, So after the 15 minute incubation with our an biotin magnetic beads I've spent on the cells and max buffer, I'm going to pour off the buffer, re suspend them in a buffer amount that's appropriate to our cell count.
So you'll have to check your milli tiny protocol for that. And then I'm going to apply them to the column that we just rinsed with max buffer. So now we're going to add the cell suspension to the column.
And because this is a negative selection column, we're going to collect the cells that flowed through and those should all be CD four positive T-cells in this case. So now I'm going to wash the column with 12 mils of max buffer, and again, we're collecting the eluting, which should contain only CD four positive T cells. So now I'm going to spin down the CD four positive T cells, resuspend them, and then label them with the appropriate dye that I'm going to use for tau imaging.
I'm also going take a small sample of the cells that I've purified and label them with anti CD three and anti CD four and run them on facts to make sure that I have a clean population of CD four positive T cells. So here's a look at the cells that we just spun down. This should be a population of pure CD three, CD four positive T cells.
So I've just shown you negative selection for CD four positive T cells. Using the Milit tiny biotech kit, we're going to adoptively transfer these cells into a mouse and use it for one of our own T Photon and machine experiments. Good luck with your own experiments.
