Isolation of Mouse Kidney-Resident CD8⁺ T cells for Flow Cytometry Analysis

All animal experiments performed following this protocol must be approved by the respective institutional Animal Care and Use Committee (IACUC). All procedures described here have been approved by IACUC UT Health San Antonio.

1. Adoptive transfer of P14 T cells into C57BL/6 recipients and LCMV infection

1. Use P14 mice at 6-12 weeks of age. Ensure that the sex of donor P14 TCR transgenic mouse should match the sex of C57BL/6 recipient mice. Otherwise, cells such as male T cells transferring into female mice will result in immune-mediated rejection of circulating donor T cells in around 12 days¹⁴.
2. Purify naïve CD8⁺ T cells from the spleen and lymph nodes of a P14 TCR transgenic mouse using mouse CD8⁺ T cell purification kit following the manufacturer’s protocol.
   1. Briefly, dissect spleen and lymph nodes, mash the samples to generate single cell suspension.
   2. To enrich for naïve T cells, add biotin-anti-CD44 antibody during the first antibody incubation step¹³.
      NOTE: Negative selection commercial kit used in this step contains two major components for two steps of incubation (i.e., biotin-antibody mixture incubation and streptavidin-magnetic bead incubation).
3. Use a hemocytometer to count the cells and inject 1,000 to 10,000 purified P14 T cells in 200 µL of PBS into each sex matched C57BL/6 recipient via the tail vein.
   NOTE: Detailed tail vein injection protocol is described in step 2.
4. On the next day, all C57BL/6 recipients receive 2 x 10⁵ pfu LCMV Armstrong diluted in 200 µL of PBS via an intraperitoneal route.

2. Intravascular labeling of CD8⁺ T cells

NOTE: Depending on the research interests, different time points can be chosen following infection. An example of day 30 post infection (day 30 after step 1.4) is demonstrated, which is often considered as an early memory time point for CD8 T cell response.

1. Dilute biotin anti-CD8α antibody to 15 µg/mL in PBS. Ensure each mouse receive 200 μL of PBS containing 3 µg antibody.
   NOTE: Biotin anti-CD8 antibody is used here so that it will be more flexible to design the final FACS staining panel. Fluorescent dye-labeled antibody can be used directly. However, it is important to use different clones of monoclonal antibodies for intravenous labeling versus FACS staining.
2. Properly heat the tail vein of mice with an overhead heat lamp for 5-10 min to dilate the veins.
   NOTE: Extra care must be taken to prevent overheating the animals. Reduce the heat or remove the heat lamp if the mice stopped to move around.
3. Draw 200 µL of prediluted anti-CD8α antibody mix into a 100 U insulin syringe (28G) and remove any air bubbles by moving the piston up and down. Bend the needle to create a 150° angle between the needle and syringe, bevel up, so the needle will be parallel to the vein.
4. Restrain the mouse with a rodent restrainer of appropriate size and spray the tail with 70% ethanol to make the vein clearly visible.
   NOTE: The duration of the restraint should be kept to a minimum.
5. Hold the tail at the distal end with thumb and middle fingers of the non-dominant hand. Place the index finger underneath the site where the needle will be inserted.
6. Hold the syringe with the dominant hand and insert the needle into the vein in parallel towards the direction of the heart.
   NOTE: When placing correctly, the needle should move smoothly into the vein.
7. Slowly inject 200 µL of anti-CD8α antibody via the tail vein.
   NOTE: If there is a resistance or a white area is seen above the needle on the tail, the needle is not inside the vein. Start the initial injection close to the tip of the tail. When necessary, move forward towards the bottom of the tail for subsequent injections.
8. Remove the needle and gently apply compression until bleeding stops.
9. Return the mouse to the cage, and euthanize the mouse after 3-5 min.
   NOTE: The mice should be euthanized via isoflurane chamber followed by cervical dislocation. Other methods, such as CO₂ inhalation will take up to 5 min and may introduce unnecessary variation of intravenous antibody labeling time.
10. Dissect the kidney using scissors, transfer the whole kidney to 1.5 mL of microcentrifuge tubes and leave the samples on ice until further processing.
    NOTE: Intact whole kidney can be safely stored on ice for a few hours before subsequent processing and digestion.

3. Enzymatic digestion of the kidney

1. Prepare 6 well plates containing 3mL of the digestion solution (RPMI/2% FCS/1 mg/ml Collagenase B) for each mouse, store on ice.
   NOTE: Stock collagenase B solution at higher concentration (e.g., 20x) can be prepared and stored at or below -20°C. Working solution should be prepared fresh. Please see Table 1 for the recipes for all solution/buffer used in the current protocol.
2. Add 300 μL of digestion solution into the 1.5 mL microcentrifuge sample tubes and mince the kidney samples with a straight spring scissor.
   NOTE: The kidney tissue should be minced into even pieces with sizes no larger than 1.5 mm in diameters. No decapsulation step is required.
3. Transfer the minced kidney samples to 6 well plates containing the digestion solution
   NOTE: 6 well plates are used for convenience only when there are multiple samples to be processed.
4. Incubate the samples at 37 °C with gentle rocking (around 60 rpm) for 45 min.
5. After digestion, mash the tissue with the plunger flange of a 3 mL syringe directly inside the dish, and transfer into 15 mL conical tubes.
   NOTE: Usually filtration through a cell strainer is not required at this step because density gradient centrifugation is performed next to purify live lymphocytes.
6. Spin down the samples at 500 x g for 5 min at 4 °C.
7. Resuspend the pellet in 3 mL of RPMI/10% FCS.

4. Density gradient centrifugation to enrich lymphocytes from the digested kidney

1. Spin down the sample at 500 x g for 5 min at 4 °C.
2. Remove the supernatant and resuspend the cell pellet with 5 mL of 44% Percoll/RPMI mix (44% Percoll + 56% RPMI without FBS).
3. Put the tip of a 3 mL pipette containing 3 mL of 67% Percoll/PBS (67% Percoll + 33% PBS) directly to the bottom of each tube, and slowly release the solution so that the heavy solution (67% Percoll/PBS) forms a distinct layer at the bottom and the light solution (44% Percoll/RPMI) is raised up. Together, cocktail layers will form.
   NOTE: For newly arrived density gradient medium from the vendor, add 1/10 volume of sterile 10x PBS to the bottle, mix well and consider the PBS-balanced density gradient medium as 100% solution.
4. Spin the samples at 900 x g for 20 min at room temperature with reduced accelerator and brake setting.
   NOTE: For centrifuges with accelerator and brake settings (on a 1-9 scale (1 means minimum and 9 means maximum)), use 6 for accelerator and 4 for brake.
5. Carefully remove the tubes from the centrifuge without disturbing the layers;
   NOTE: A clear lymphocyte layer is identified between pink color RPMI layer and colorless PBS layer.
6. Remove the top layer with a transfer pipette without touching the lymphocyte layer.
7. Transfer the lymphocyte layer to a new 15 mL tube, fill the tube with PBS/5% FCS and mix by inverting the tube 4-6x slowly to ensure sufficient mixing.
   NOTE: This step is important to wash out any residual Percoll.
8. Spin down the samples at 500 x g for 5 min at 4 °C.
9. Remove the supernatant and re-suspend the cell pellet with 500 μL of complete RPMI medium. The cells are ready for flow cytometry staining.

5. Flow cytometry staining and analysis

NOTE: Please follow standard flow cytometry staining protocol for T lymphocytes.

1. Take around 1 x 10⁶ cells for each FACS staining.
   NOTE: Use a U-bottom 96 well plate for FACS staining. However, if only a limited number of samples are involved, 5 mL FACS tubes can also be used.
2. Spin down the cells at 500 x g for 5 min at 4 °C. Discard the supernatant.
3. Resuspend each sample in 50 µL of FACS buffer containing 10 µg/mL of 2.4G2 FcR blocker (an anti-CD16/32 monoclonal antibody to block the interaction between added FACS antibody with FcR). Incubate on ice for 15 min.
4. Without further centrifugation, add 50 µL of surface staining antibody mixture for each sample so that the final staining volume is 100 µL/each sample. Incubate on ice for 30 min in the dark.
   NOTE: Include fluorescently labeled streptavidin (2 µg/mL or follow manufacturer’s recommendation) in the antibody mixture to label CD8α⁺ intravascular CD8⁺ T cells and use fluorescent labeled anti-CD8β antibody to label all CD8 T cells. Antibody mixture is made by adding desired amounts of interested antibodies into the FACS buffer.
5. Wash the samples with PBS twice. For each wash, fill the wells of 96 well plate with cold PBS, spin at 500 x g for 5 min at 4 °C and discard the supernatant.
6. Resuspend the cells in 100 µL/well of diluted live/death dye. Incubate on ice for 30 min in the dark.
   NOTE: Even with density gradient medium spin, enzymatic digestion step often induces significant cell death in tissue-resident T cells due to ARTC2.2/P2RX7 signaling¹⁵. Live/death dye staining is highly recommended before fixation. Anti-ARTC2 nanobody can be administrated into the mice before euthanasia to prevent cell isolation-induced cell death.
7. Wash the samples with FACS buffer twice. For each wash, fill the wells of 96-well plate with cold FACS buffer, spin at 500 x g for 5 min at 4 °C and discard the supernatant.
8. Resuspend the cells in 100 μL/well fixing buffer. Incubate at 37 °C for 10 min.
   NOTE: Fixing buffer is made freshly by diluting formaldehyde with PBS to a final concentration of 2% formaldehyde.
9. Wash the samples with FACS buffer twice. For each wash, fill the wells of 96 well plate with cold FACS buffer, spin at 500 x g for 5 min at 4 °C and discard the supernatant.
10. Resuspend the cells in 250 μL/well FACS buffer. Seal the plate, store at 4 °C and protect from light until FACS analysis.
    NOTE: Before FACS analysis, filter the samples through a 70 μM nylon mesh to remove possible cell clusters that may clog the FACS machine.