In Vitro Generation of Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors

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

1. Preparation of AC-6 Feeder cells

Note: The AC-6.21 (AC-6 in short) stromal cell line (provided by I. Weissman, Stanford University) should be maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS). To serve as feeder cells, AC-6 cells are γ-irradiated with 3,000 rad (30Gy) one day before the co-culture to prevent their proliferation. Note that AC-6 cells tend to lose their differentiation-supporting ability if left overcrowded during maintenance, or if their passage number is over 20.

1. Wash AC-6 cells once with 1 ml Dulbecco's Phosphate-Buffered Saline (DPBS) and remove DPBS by aspiration. Treat AC-6 cells with 0.8 ml trypsin solution (0.05 % trypsin and 0.5 mM EDTA in DPBS) for 3-5 min at 37^oC, 5% CO₂ and then stop the reaction by diluting it with 10 volumes of culture medium to make single cell suspension.
2. Seed AC-6 cells at 5.9×10⁴ cells/well into 12-well plates and incubate them at 37^oC, 5% CO₂ O/N.
   Note: The density of AC-6 cells is very important as a lower density will favor cDC generation. Seeding at a density of 5.9×10⁴ cells/well will allow AC-6 to reach confluency by the next day, which is optimal for the derivation of pDCs from CLPs.
3. Irradiate AC-6 cells at 3,000 rad (30 Gy) using γ-irradiator.
   Note: The dose used to irradiate AC-6 is optimized to prevent the cells from proliferating and yet keep them viable long enough to provide the cytokines and cell-cell contact required for the differentiation of DCs from CLPs.
4. Aspirate medium and replace with 1 ml complete RPMI (RPMI with 10% heat-inactivated FBS, 50 µg/ml gentamycin and 50 µM β-mercaptoethanol).

2. Isolate BM Cells from Mice

1. Sacrifice mice by CO₂ asphyxiation and cervical dislocation. Place the mice on a dissection tray and sterilize them with 70% ethanol. Make an incision at the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.
2. Dissect mice in a semi-sterile hood. To release the femur and tibiae, clip the femur above and the tibiae below the knee joint. Detach the muscles from the bones using scissors and place the bones in a 6 cm petri dish containing 5 ml complete RPMI.
3. Move to a sterile culture hood. Fill a 3 ml syringe connected to a 27G needle with complete RPMI. Cut off both ends of the bones using scissors, and insert the 27G needle to flush the marrow out of the bones by gently injecting complete RPMI once from each end.
4. Prepare a single-cell suspension by gently pipetting the cells up and down 3-5 times in the culture dish using the syringe without the needle.
5. Centrifuge the cells for 5 min at 500 x g and decant the supernatant.
6. Lyse red blood cells with 1 ml ACK buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) incubating for 1 min and stopping the reaction by diluting with 10 volumes of complete RPMI. Allow cells to stand for 5 min in order to pellet down dead cell clumps and tissue debris by gravity.
   Note: Do not stand for more than 5 min as this will result in cell loss due to the settling of viable cells.
7. Slowly decant cell-containing supernatant to a new tube leaving debris behind in the original tube.
8. Centrifuge for 5 min at 500 x g to pellet the cells, then remove and discard the supernatant.
9. Resuspend total BM cells (typically 4-6 x 10⁷ BM cells are obtained from one mouse) in 100 µl FACS buffer (1x PBS+ 2% FBS+ 1mM EDTA).

3. FACS Analysis and Sorting of CLPs

1. Add anti-CD16/32 (hybridoma supernant clone 2.4G2, 50 µl/reaction or 1-2 µg/reaction) into cells in FACS buffer (step 2.9) for 1-2 min in order to block Fc receptors.
   Note: Anti-CD16/32 is also called Fc block, which is added to prevent the non-specific binding of antibodies to cells expressing Fc receptors including granulocytes, monocytes, and B lymphocytes.
2. Simultaneously stain cells with the following fluorescent dye-labeled antibodies (for 4×10⁷ cells) for 15 min on ice, avoiding ambient light. Antibodies: PE-conjugated lineage markers (0.2 µg each) including anti-CD3 (17A2), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), and anti-MHC-II (NIMR-4), 0.2 µg anti-c-Kit-PerCP-Cy5.5 (2B8), 1 µg anti-Sca-1-FITC (D7), 0.4 µg anti-M-CSFR-APC (AFS98), and 0.2 µg anti-IL-7Rα-PE-Cy7 (A7R34).
3. Wash cells with 3 ml FACS buffer, and centrifuge for 5 min at 500 x g.
4. Resuspend the cells in 300 µl FACS buffer and filter cells through a 40 µM cell strainer.
5. Wash the tube with an additional 100 µl FACS buffer to recover any remaining cells and filter into a sterile FACS sorting tube using the same cell strainer.
6. Perform flow cytometric analysis immediately after the staining using appropriate filters and voltages for signal detection. Use a 488 nm laser to detect the FITC-, PerCP-Cy5.5-labeled antibodies, a 561 nm laser to detect the PE- and PE-Cy7-labeled antibodies, and a 633 nm laser to detect the APC-labeled antibody.
7. Sort out CLPs according to the following markers lin^–c-kit^intSca-1^intM-CSFR-IL-7Rα⁺ (as stained in step 3.2) using a cell sorter. Collect the sorted cells in a 15 ml tube containing 8 ml of complete RPMI as a cushion.
8. Record the absolute number of sorted cells as shown by the cell sorter at the end of the run. Typically, 5×10⁴ CLPs can be obtained from the BM of one mouse.

4. Coculture CLPs and AC-6

1. Centrifuge the sorted cells from step 3.7 for 10 min at 500 x g, remove the supernatant, and resuspend the cell pellet with enough complete RPMI to obtain a cell density of around 5×10⁴ cells/ml. Seed 500 cells/well into the 12-well plate containing feeder cells prepared in step 1.4.
2. Add 100 ng/ml FL (hu-Flt3L-Ig generated in-house using an expression system provided by M. Manz, University Hospital Zürich, Switzerland) and incubate at 37^oC, 5% CO₂ with periodic visual monitoring of DC development under a microscope.
   Note: Commercially available recombinant human FL or mouse FL can be used as a substitute for hu-Flt3L-Ig to support DC development.
3. Collect the cells in the supernatant on day 12 and wash the wells once with 0.5 ml complete RPMI medium combining the resulting supernatants. Add 0.5 ml fresh medium and scrap the adherent cells with a cell scraper.
4. Combine the cell-containing medium from both parts and centrifuge for 5 min at 500 x g.
5. Decant supernatant and resuspend cells in 50 µl FACS buffer. Add 50 µl anti-CD16/32 hybridoma supernatant and incubate for 1-2 min to block Fc receptors.
6. Enumerate and stain all the cells with the following antibodies (0.05 µg each) anti-CD11c-APC (N418), anti-CD11b-FITC, and anti-B220-PE.
7. Wash and centrifuge the cells as described in step 3.3.
8. Resuspend the cells in 100 µl FACS buffer, gate on CD11c+, and analyze for cDCs (CD11c⁺CD11b⁺B220^–) and pDCs (CD11c⁺CD11b^–B220⁺).