The overall goal of this procedure is to describe how to isolate pure and double negative T cells from the mouse kidney. This is accomplished by first digesting the kidney in a collagenase solution. Next, the mononuclear cells are separated by density gradient, and hematopoietic CD 45 positive cells are collected by positive selection.
Ultimately, the double negative T cells can then be isolated by negative selection. In 1998, our team found that immune cells, particularly T cells, contrary to current dogma were mediating acute kidney injury. There were very few of those cells in the kidney and heart to study, so we developed a technique to better study those.
We were surprised to find a population of cells, the double negative T T-cell without CD four or CD eight that were in high population, both in normal kidney and during injury. The detailed and improved technique to study these better and to look into the mechanisms will be described in this upcoming video. We have been studying double negative T cells since 2001, and recently we joined hand with Dr.Rob lab to modify the regional method for isolation of these cells to get a pure population that can be used for different biological immune responses.
Although this method can provide an insight into the immune cells in the kidney, it can also be applied to other systems such as non lymphatic tissues Before starting the surgery. First, add 10 milliliters of collagenase D to a Petri dish for tissue collection. Then spray the abdominal area of a euthanized mouse with 70%ethanol.
Next, make a midline incision through the abdominal skin and peritoneum from the sternum to the pubis, and then move the intestine laterally to the side to expose the left kidney. Identify the left capsule, which appears as a transparent layer surrounding the kidney and is partially covered by perinephric adipose tissue and carefully separated from the kidney with forceps. Then cut the pedicle vessel with surgical scissors to remove the tissue, place the kidney into the Petri dish.
And then after removing the right kidney, use a regular metal shaping blade to cut the kidney tissue into small one to two millimeter pieces. Digest the pieces in the collagenase solution for 30 to 45 minutes at 37 degrees Celsius, and then to isolate the kidney mononuclear cells mechanically disrupt the kidney slurry through a 70 micron strainer. Resus suspend the cell suspension in 25 milliliters of tissue culture medium.
And then after gentle tation, spin down the cells and resuspend the pellet in four milliliters of 40%per call solution. Next, use a plastic transfer pipette to gently overlay the per call cell solution onto four milliliters of an 80%per call solution to create two phases clearly separated by a translucent layer. Separate the cells for 30 minutes at 1500 Gs and room temperature with the break off, and then asbury about one to two milliliters of the thick yellow lipid containing top layer.
Use a new transfer pipette to collect the slightly whitish translucent layer from the interface of the two phases into a 15 milliliter conical tube containing two milliliters of tissue culture medium. Then add 13 milliliters of medium to bring the total volume to 15 milliliters. After washing and counting, dilute the cells to one times 10 to the seven in 90 microliters of running buffer.
Then incubate the cells in 10 microliters of CD 45 microbeads at four degrees Celsius. After 15 minutes, add one milliliter of running buffer to stop the reaction. Then spin down the cells and reus.
Suspend the pellet in 500 microliters of fresh running buffer. Next, place a column into a bead separation magnet and rinse the column with three milliliters of running buffer. Then pass the cell suspension through the magnetic column, collecting the unlabeled cells that pass through the column.
Wash the tube containing the cells three times with three milliliters of running buffer, collecting the effluent each time, and then remove the column from the magnet. Pipette five milliliters of running buffer onto the column and immediately flush out the labeled fraction containing the CD 45 positive cells. Then after counting the cells, incubate them in 2.5 microliters of each of these biotinylated antibodies for every one times 10 to the seven hematopoietic cells at four degrees Celsius.
After 30 minutes, incubate the cells for another half hour at four degrees Celsius in one milliliter of anti biotin microbeads. Place a new column in the magnet and then load the cells onto the column as just demonstrated this time collecting the negative fraction. Finally, count the viable cells to determine the total number of double negative T cells in the negative fraction and multiply this number by the percent purity as determined by flow cytometric analysis.
While type C 57, black six kidneys contain approximately 1.5 to 2.1 times 10 of the six mononuclear cells per kidney, less than 10%are hematopoietic CD 45 positive cells. After density gradient centrifugation to collect the kidney mononuclear cell layer, the CD 45 positive cells can be further purified to about 80 to 95%by CD 45 positive magnetic bead separation. Among enriched hematopoietic CD 45 positive cells, about 30 to 60%are double negative T cells.
Negative selection of the CD 45 positive cells to remove the CD four positive, CD eight positive MHC class two positive and tetramer positive cells results in highly purified 90 to 95%pure double negative T cells. Using this protocol, it is possible to obtain up to 0.5 times 10 of the six hematopoietic CD 45 positive magnetically labeled cells per mouse. This procedure will provide a better understanding of the biological functions and mechanism for factions of double negative T-cells in different tos.
