A Method for the High Salt Treatment of Dendritic Cells and their Adoptive Transfer into Mice

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolation of Spleens from Mice

1. Prepare 1640 RPMI (Roswell Park Memorial institute): 10% fetal bovine serum (FBS), 0.10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 50 µM β-mercaptoethanol and 1% penicillin/streptomycin.
2. Euthanize 10–12 week-old C57bl/6 male mice by CO₂ inhalation. Spray the chest and sides of the mouse with 70% ethanol. On the left side, carefully open the skin and the peritoneal cavity to expose the spleen.
3. Using small forceps, cautiously move the intestines and liver to the left side of the mouse, and using fine scissors, gently excise the spleen.
   NOTE: This step must be done very carefully, as damage to the gastrointestinal tract can induce contamination.
4. Place each excised spleen in a labeled 15 mL conical tube containing 3 mL of RPMI 1640 medium.

2. Generation of Single Cell Suspensions from Spleens

NOTE: The spleen can be dissociated into a single-cell suspension by combining mechanical dissociation plus enzymatic digestion.

1. Prepare the spleen digestion solution by adding collagenase D (1 mg/mL; 0.29 U/mg lyophilized) and DNase I (0.1 mg/mL) to prepared RPMI 1640 medium (see step 1.1)
2. Use forceps to transfer the spleen into a C tube (dissociation tube) containing 3 mL of spleen digestion solution.
3. Perform the mechanical dissociation using a semi-automated homogenizer according to the manufacturer's instructions.
   1. Place the C tube onto the semi-automated homogenizer, ensuring that the C tube is tightly placed on the rotating arm. Once the C tube is placed, press the start button on the semi-automated homogenizer to run the Spleen 04.01 protocol for 60 s.
      ​NOTE: Mechanical dissociation can also be performed by placing a 40 μm filter on top of a 50 mL conical tube and gently grinding the spleen through the filter. After grinding the spleen, through rinse the 40 µm filter with 10 mL of RPMI media.
4. After mechanical dissociation, detach the tube from the homogenizer and perform the enzymatic digestion. Incubate the samples for 15 min at 37 °C under continuous rotation (20 rpm).
5. Transfer the solution by pipetting it into a 50 mL conical tube after filtering through a 40 µm strainer. Wash the 40 µm strainer with 10 mL of Dulbecco's PBS (dPBS). Centrifuge the cells at 300 x g for 10 min at 4 °C.
6. After centrifugation, aspirate the supernatant completely and wash the pellet by resuspending it in 10 mL of dPBS. Centrifuge at 300 x g for 10 min at 4 °C.

3. Isolation of CD11c⁺ DCs from Splenic Single Cell Suspension

1. Resuspend the cell pellet in 5 mL of dPBS and count the number of cells using a hemocytometer and trypan blue exclusion.
2. Pellet the cells by centrifuging at 300 x g, for 10 min at 4 °C.
3. Aspirate the supernatant completely and resuspend the pellet in 400 µL of magnetically activated cell-sorting (MACs) buffer.
4. Add 100 µL of CD11c microbeads (see Table of Materials) per 1 x 10⁸ cells.
5. Vortex the cell suspension and incubate for 10 min in the refrigerator at 4 °C.
6. Add 10 mL of MACs buffer to wash cells. Centrifuge at 300 x g for 10 min at 4 °C.
7. Aspirate the supernatant completely and resuspend in 500 µL of MACs buffer.

4. Magnetic Separation of CD11c⁺ DCs

NOTE: Magnetic separation is done manually with a magnetic separator (see Table of Materials) outfitted with LS Columns. Magnetic separation can also be done automatically with an automated magnetic separator. (see Table of Materials).

1. Place the LS column in the magnetic field of MACs separator and a 15 mL conical tube under each column to collect the flow-through.
2. Rinse the LS column with 3 mL of buffer.
3. Place the cell suspension onto each LS column and collect the flow-through containing unlabeled cells.
4. Wash the LS column with 3 mL of MACs buffer, collecting the unlabeled cells that pass through. Wash the LS column 2 more times.
5. Remove the LS column from the magnetic separator. Add 5 mL of MACs buffer to each column and immediately flush out the magnetically labeled cells by firmly plunging the LS column into a clean 15 mL conical tube.
   NOTE: It is important to perform a double isolation of CD11c cells to obtain a high-purity cell suspension. Therefore, repeat steps 3.1 through 4.5. and reference Figure 4 for purity standards. This step improves the purity of CD11c⁺ cells to 90–95%.
6. Determine CD11c⁺ DC number on a hemocytometer using trypan blue exclusion. Dilute the cell sample at a 1:1 dilution in 0.4% trypan blue solution.
   NOTE: Non-viable cells will be stained blue, while viable cells will remain unstained.
   1. To do this, carefully fill a hemocytometer with 10 µL of the cell sample dilution and incubate for 1 minute.
   2. Count cells in 4 squares of 1 x 1 mm² in the chamber that has been loaded to determine the viable cell number.

5. In Vitro High Salt Treatment of CD11c⁺ DCs

1. Prepare DC culture medium by adding 10% FBS, 0.1 mM HEPES, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol and 1% penicillin/streptomycin to 500 mL 1640 RPMI.
2. Prepare 10x DC high salt cell culture media (400 mM) by weighing out 1.17 g of NaCl and placing it into a sterile 50 mL falcon tube. Add 50 mL of sterile DC cell culture media (prepared in step 5.1) to the 50 mL falcon tube and vortex until the NaCl is in solution.
3. Immediately filter the high salt DC culture media using vacuum filtration through a 0.2 μm filter in a cell culture hood.
4. Centrifuge each cell suspension from the spleens at 300 x g for 10 min at 4ºC. Resuspend each cell pellet in DC culture media at a concentration of 1 x 10⁶ cells/mL.
5. Pipette 900 μL (approximately 1 x 10⁶ cells) of the DC suspension in a 24-well flat bottom Falcon plate. Add 100 µL of high salt DC culture media to each well for a final sodium concentration of 190 mM. Label the plate and place in a 37 °C humified CO₂ incubator for 48 h.

6. Adoptive Transfer of High Salt Treated CD11c⁺ DCs

1. After in vitro stimulation for 48 h, remove the plate from the 37 °C humified CO₂ incubator.
2. Pipette the cell culture media up and down to resuspend the CD11c⁺ DCs. Pipette all the CD11c⁺ DC suspension into a corresponding labeled 1.6 mL tube. Repeat this step for each individual well plated.
3. Centrifuge each CD11c⁺ DC suspension at 300 x g for 10 min at 4 °C. Aspirate the supernatant. Resuspend the DC pellet with 100 µL of sterile dPBS.
4. Draw DC suspension into a 1 mL syringe equipped with a 27 G ½ inch needle.
5. Place the naïve male 10-week-old C57bl/6 mice under 2% isoflurane to achieve a stable surgical plane. Check the level of anesthesia with the lack of pedal reflex. Once a stable surgical plane is achieved, slowly and carefully introduce the needle into the retro-orbital space at an angle of approximately 30°.
   NOTE: The bevel of the 27 G needle should be pointing down, away from the eye, to prevent ocular damage.
6. Slowly and smoothly inject the CD11c⁺ DC suspension (approximately 1 x 10⁶ cells). Once the injection is complete, carefully remove the needle from the retro-orbital space being conscious not to damage the eye.
   NOTE: After the injection, there should be little or no bleeding. Adoptive transfer of CD11c⁺ DCs can also be performed using an I.V. method of injection (e.g. Tail vein). The control mice received CD11c⁺ DCs that were cultured in normal salt media.
7. Remove the mice from the nose cone and monitor them for approximately 30 min during the recovery from anesthesia.