Isolation of Double Negative αβ T Cells from the Kidney

1. Preparation of Instruments, Culture Media and Reagents

1. Instruments: Prepare the reagents under sterile conditions and use under a laminar flow hood.
2. Prepare 1 L of RPMI tissue culture medium, add 5% of fetal bovine serum, 2.1 g of bicarbonate, 10 ml glutamate 100x, 10 mg of HEPES, 1 mg sodium pyruvate and 10 ml of 100x penicillin/streptomycin.
   Note: the recommended tissue culture medium, RPMI, is interchangeable with DMEM.
3. Prepare 5% collagenase “D” solution (5 ml of collagenase “D” to 9 ml of tissue culture medium).
4. Make Percoll solution at three different concentrations: 100%, 80% and 40%. The following concentrations are calculated for 1 sample. Keep solutions at room temperature.
   1. Percoll solution: add 9 ml of undiluted Percoll (from stock solution); add 1 ml of PBS 10x.
   2. Percoll solution: add 8 ml of Percoll 100%; add 2 ml of PBS 1x.
   3. Percoll solution: add 4 ml of Percoll 80%; add 4 ml of PBS 1x.
5. Make 1 L of fluorescence-activated cell sorting (FACS) buffer; add 5 ml 10% BSA (0.1% BSA), 1 ml 10% sodium azide (0.1%), 2 ml EDTA agent, make up to 1,000 ml with 1x PBS.

2. Preparation of Kidney Digestion

1. Having acquired any necessary institutional approval, sacrifice and exsanguinate the mice with standard methods and following the current regulations and animal care guidelines.
   1. Anesthetize the mouse with pentobarbital (0.07 mg/kg).
   2. Proceed to exsanguination.
   3. Place the mouse in a supine position on a surgical table.
   4. Spray the abdominal area with 70% ethanol.
2. Add 10 ml of collagenase D solution to the Petri dish.
3. Remove and decapsulate both kidneys from each mouse.
   1. Open the abdomimal cavity by making a midline incision through the abdominal skin and peritoneum from the sternum to the pubis.
   2. Expose the left kidney by moving the intestine laterally to the side.
   3. Carefully separate the left capsule with the forceps.
      Note: The capsule appears as a transparent layer surrounding the kidney and partially covered by perinephric adipose tissue.
   4. Remove the left kidney by cutting the pedicle vessels using a surgical scissor.
   5. Repeat same procedure for the right kidney.
      Note: The right kidney is more cranial and closer to the midline (pedicels are shorter).
4. Place the kidney into the Petri dish.
5. Cut the kidney tissue into small pieces (1-2 mm in dimension) using a regular metal shaping blade and place the pieces into the collagenase solution for 30-45 min in the incubator at 37 °C (Figure 1).

3. Preparation of Kidney Mononuclear Cells (KMNC) from the Kidney Digestion

1. Obtain a cell suspension of the kidney digestion by mechanically disrupting the tissue using a strainer (70 µm) and resuspend in 25 ml of tissue culture medium and mix.
2. Centrifuge at 400 x g for 10 min at 4 °C to pellet the cell suspension.
3. Re-suspend the pellet in 4 ml of 40% Percoll solution and gently overlay onto 4 ml of 80% Percoll solution with a transfer plastic pipette, very slowly and carefully. This will result in two phases clearly separated by a translucent layer. On the top will be a yellow layer corresponding to the lipids.
4. Centrifuge at 1,500 x g for 30 min at room temperature with the centrifuge in brake off mode.
5. Remove by aspiration the yellow and thick top layer (about 1-2 ml).
6. Collect only the slight whitish translucent layer from the interface of the two phases with a transfer pipette. Very carefully transfer this suspension into a 15 ml conical tube containing 2 ml of tissue culture medium and then add 13 ml of medium to make a total volume of 15 ml.
7. Centrifuge at 400 x g for 10 min at 4 °C.
8. Resuspend the samples in 1 ml of the medium.
9. Count the number of KMNC using trypan blue exclusion on a hemocytometer and express the results as numbers of KMNC per ml per Kidney.
10. Wash once as above.

4. Isolation of Hematopoietic (CD45+) Cells from KMNC (Step I)

1. Resuspend the cells up to 10⁷ into 90 µl of running buffer.
2. Add 10 µl of CD45 microbeads and incubate for 15 min at 4 °C.
3. Add 1 ml of the running buffer to stop the reaction. Centrifuge at 400 x g for 10 min at 4 °C and resuspend in 500 µl of running buffer.
4. Prepare the magnetic column by placing at the magnet and rinse with 3 ml of running buffer.
5. Pass the cell suspension through the magnetic column and collect the unlabeled cells that pass through the column.
6. Wash the cells three times with 3 ml of running buffer. Then collect the effluent.
7. Remove the column from the separator and pipette 5 ml of running buffer onto the columns and flush out the labeled fraction immediately. This fraction contains the CD45+ cells. Count the cells.
8. To calculate the absolute number of different T cells subsets into kidneys multiply the total number of KMNC by the percentage of positive cells determined by flow cytometry (Figure 2).

5. Isolation of DN T Cell from CD45+ Preparation by Negative Selection (Step II)

1. Add 2.5 µl (0.5 mg/ml, 1.25 µg) of each of the following biotinylated antibodies: anti-CD4, anti-CD8, anti-Fc receptor (CD16/32), anti-MHC class II (I-A b), anti-CD1d PBS-57 tetramer for every 10⁷ hematopoietic cells.
2. Incubate cells for 30 min in cold room.
3. Add 1 ml anti-Biotin microbeads.
4. Incubate for 30 min in a cold room.
5. Place the tube in the magnet and proceed to depletion.
6. Count the viable cells to determine the total number of DN T cells in the negative fraction by using a hemocytometer.
7. Check purity of DN T cells with flow cytometry by following this gating strategy: first gate CD45+, second gate TCRαβ+. Exclude CD1d+ cells. Plot CD4+ vs. CD8+ to identify DN T cells (Figure 3).
8. Multiply the total number by the percentage determined in purity.
   Note: Using this protocol results in a population of more than 90% pure DN T cells.