Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) 

1. Preparation of Reagent
   1. Prepare PBS/EDTA: phosphate-buffered saline solution (PBS) and supplement with 2 mM ethylenediaminetetraacetic acid (EDTA). Sterilize this solution by filtration through a 0.2 µm filter. NOTE to store PBS/EDTA at 4 °C and warm to room temperature before use.
   2. Prepare staining buffer: phosphate-buffered saline solution (PBS) supplement with 2% fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 2 mM ethylenediaminetetraacetic acid (EDTA). Sterilize this solution by filtration through a 0.2 µm filter.
2. Collect Blood from Blood Cone
   Note: The blood cone contains white blood cell components collected after plateletpheresis from the hospital. If blood is collected in heparin or EDTA tubes, dilute the blood with PBS in a 1:1 ratio and proceed to step 1.3; if a buffy coat is received, dilute the buffy coat with PBS in a 1:2 ratio and proceed to step 1.3.
   1. Cut the two ends of the cone to allow blood to flow out into a 50 ml tube.
      Note that the cone usually contains 10 ml of blood.
   2. Use a blunt-end syringe containing 30 ml of PBS/EDTA to wash the cone and collect it in a 50 ml tube.
   3. Dilute blood further with PBS/EDTA to a final volume of 80 ml.
3. Isolation of PBMCs by Density Centrifugation
   1. Aliquot 15 ml Ficoll each to 4 fresh 50 ml tubes.
   2. Use a 25 ml serological pipet to add 20 ml of diluted blood over the Ficoll layer. Take note to hold the 50 ml tube at a 45 angle and take care to not disturb the interphase.
   3. Centrifuge the tubes at 805 x g without brakes for 30 min, 20 °C.
   4. Remove the plasma layer and collect the ring of PBMCs lying just below the plasma layer with a Pasteur pipet. Combine four tubes of PBMCs into two 50 ml tubes. Note to avoid collecting the transparent layer below the PBMCs.
   5. Add PBS/EDTA to a final volume of 50 ml per tube of PBMCs and centrifuge at 548 x g with brakes for 10 min, 20 °C.
   6. Aspirate supernatant and resuspend pellet in each tube with 25 ml of staining buffer and combine into one 50 ml tube.
   7. Centrifuge at 367 x g with brakes for 5 min, 4 °C.
   8. Aspirate supernatant and resuspend pellet with 10 ml of staining buffer.

2. Monocyte Enrichment by Magnetic Separation

1. Determine Cell Number
   1. Take 20 µl of PBMC cell suspension and mix it with 20 µl of trypan blue to count the number of live cells using a cytometer.
   2. Centrifuge cell suspension at 367 x g with brakes for 10 min, 4 °C.
   3. Aspirate supernatant completely and resuspend cell pellet to a concentration of 10⁷ cells per 80 µl of staining buffer.
2. Magnetic Labeling
   1. Add 20 µl of CD14 microbeads per 10⁷ cells, mix well, and incubate for 15 min at 2 – 8 °C.
   2. Wash cells by adding 1 ml of staining buffer per 10⁷ cells and centrifuge at 367 x g with brakes for 10 min, 4 °C.
   3. Aspirate supernatant completely and resuspend the cell pellet in a concentration of 10⁷ cells per 50 µl of staining buffer.
3. Magnetic Separation
   1. Use medium size column for a maximum of 2 x 10⁸ total cells.
      NOTE: Choose an appropriate column and separator according to the number of total cells and the number of CD14⁺ cells recommended in the datasheet.
   2. Place the column in the magnetic field of a suitable separator and prepare the column by rinsing it with 500 µl of staining buffer.
   3. Pipette cell suspension onto the column and collect unlabeled cells that pass through the column with a 15 ml tube.
   4. Replace a new 15 ml tube under the column and wash the column 3 times with 500 µl of staining buffer. Make sure the column reservoir is empty before adding a new staining buffer between washes.
   5. Remove the column from the separator and place it on a fresh 15 ml tube.
   6. Pipette 1 ml of staining buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger (provided in the kit) into the column.
   7. Repeat steps 2.3.2 to 2.3.6 using the eluted fraction on a new column to increase the purity of CD14+ cells. Note that the beads will be released from the cells automatically in culture during step 3.2.

3. Differentiation of Dendritic Cells to Different Activation States 

1. Preparation of Reagent (Endotoxin Level has to be Less than 0.1 EU/ml in all Reagents)
   1. Prepare cell culture medium: RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% Non-Essential Amino Acids (NEAA), and 0.05 mM 2-mercaptoethanol (2ME). Sterilize this solution by filtration through a 0.2 µm filter.
   2. Prepare cytokines: reconstitute IL-4 and GM-CSF in cell-culture grade water to a concentration of 0.25 mg/ml respectively under aseptic conditions. Aliquot cytokines into 200 µl microcentrifuge tubes and store them at -80 °C.
   3. Prepare vitamin D3 stock: reconstitute vitamin D3 in cell-culture grade water to a concentration of 100 mM under aseptic conditions. Aliquot vitamin D3 into 1.5 ml microcentrifuge tubes and store at -20 °C.
   4. Prepare dexamethasone stock: reconstitute dexamethasone in cell-culture grade water to a concentration of 10 mM under aseptic conditions. Aliquot dexamethasone into 1.5 ml microcentrifuge tubes and store at -20 °C.
   5. Prepare LPS: reconstitute Lipopolysaccharides (LPS) in cell-culture grade water to a concentration of 1 mg/ml under aseptic conditions. Aliquot LPS into 1.5 ml microcentrifuge tubes and store at -20 °C.
2. Generating Different moDCs
   1. Seed 4 sets of CD14⁺ monocytes in the concentration of 0.3 – 0.5 x 10⁶/ml of cell culture medium supplemented with 200 ng/ml of GM-CSF and 200 ng/ml of IL-4 in 6 well plates. Note that this is Day 0 and the volume of medium in each 6 well is 2 ml.
   2. Incubate cells in a tissue culture incubator at 37 °C with 5% CO₂.
   3. Remove 850 µl of medium from the culture at Day 4 and centrifuge at 300 x g for 5 min, 4 °C. Aspirate supernatant and resuspend pellet in 1 ml of cell culture medium containing 2x of (200 ng/ml of GM-CSF and 200 ng/ml of IL-4). Add this cell mixture back to the culture. Note that the 2x concentration added for GM-CSF and IL-4 will become 1x when the 1 ml of medium is added back into the culture.
   4. Add 1 µl of vitamin D3 stock and 1µl of dexamethasone stock per ml of medium to two of the sets on Day 5 to generate tolerogenic moDCs. Note that the final concentration of vitamin D3 is 100 nM and dexamethasone is 10 nM; the addition of GM-CSF and IL-4 is not required on Day 5.
   5. Add 200 ng/ml of GM-CSF and 200 ng/ml of IL-4 to all the sets on Day 6.
   6. Add 1 µg/ml of LPS to one of the sets treated with only GM-CSF and IL-4 at Day 6 to generate mature moDCs.
   7. Add 1 µg/ml of LPS to one set of the tolerogenic moDCs on Day 6 to generate LPS-tolerogenic moDCs.
   8. Harvest the different types of moDCs on Day 7 by flushing the culture dish with PBS, EDTA for flow cytometry or other studies. Note that only non-adherent cells are harvested. Take note that the percentage yield from CD14⁺ monocytes for immature moDCs, mature moDCs, tolerogenic moDCs, and LPS-tolerogenic DCs are about 90%, 50%, 60%, and 60% respectively, and varies between blood donors and FBS lot.