Dendritic cells (DCs) are potent antigen-presenting cells and key regulators of the immune response. These cells are of great interest for research in cancer immunotherapy, vaccines, and infectious diseases. The protocol below describes how to generate monocyte-derived DCs (Mo-DCs) using the animal component-free (ACF) ImmunoCult™ Dendritic Cell Culture Kit. Mature DCs generated from monocytes in 7 days are functional and ready for downstream applications. This protocol can also be found in the ImmunoCult™ Dendritic Cell Culture Kit (Catalog #10985) Product Information Sheet.

Materials

• Monocytes intended for differentiation
• ImmunoCult™ Dendritic Cell Culture Kit (Catalog #10985)
• Cultureware (96-well plate, 24-well plate, 12-well plate, or 6-well plate; T-25 cm2 flask)

Protocol

Please read the entire protocol before proceeding. The following instructions are for use with a T-25 cm² flask. If using alternative cultureware, adjust volumes accordingly. This protocol is intended for use with monocytes isolated from fresh (< 24 hours old) human whole blood or from leukapheresis samples using an EasySep™ negative selection cell separation kit.

1. If using a T-25 cm² flask, add purified human monocytes at 1 x 10⁶ cells/mL to 5 mL of ImmunoCult™ DC Differentiation Medium.
2. Refer to Table 1 for volumes required for other types of cultureware.

   Table 1. Recommended Volumes of ImmunoCult™ DC Differentiation Medium for Various Cultureware

   Cultureware

   Volume of ImmunoCult™-ACF Dendritic Cell Medium

   Number of Cells/Well

   96-well plate

   100 µL/well

   1 x 10⁵

   24-well plate

   500 µL/well

   5 x 10⁵

   12-well plate

   1 mL/well

   1 x 10⁶

   6-well plate

   2.5 mL/well

   2.5 x 10⁶
3. Add the cell suspension (5 mL) to a T-25 cm2 flask. Incubate at 37°C for 3 days.
4. Day 3: Remove the medium by pipetting from the flask and add to a 15 mL conical tube (e.g. Catalog #38009). Quickly, add 5 mL of fresh ImmunoCult™ DC Differentiation Medium to the culture flask.
5. Centrifuge the tube containing medium and cells (from step 4) at 300 x g for 10 minutes. Remove and discard the supernatant. Resuspend cells in a small volume (i.e. 50 µL or up to 10% of the original volume) of fresh ImmunoCult™ DC Differentiation Medium and return to the culture flask.
   NOTE: This step saves non-adherent or loosely adherent cells.
6. Incubate at 37°C for 2 days.
7. Day 5: Thaw ImmunoCult™ Dendritic Cell Maturation Supplement at room temperature (15 - 25°C) or at 37°C until just thawed. Mix thoroughly.
   NOTE: If necessary, centrifuge supplement for 30 seconds to collect liquid from cap.
   NOTE: If not used immediately, aliquot and store at 2 - 8°C for up to 1 month. Alternatively, store aliquots at -20°C. Do not exceed the shelf life of the supplement. After thawing aliquots, use immediately. Do not refreeze.
8. Add Maturation Supplement directly to the culture flask at a 1 in 100 dilution (e.g. add 50 µL Maturation Supplement to approximately 5 mL culture medium). Swirl gently to mix. Do not change medium.
9. Day 7: Harvest fully mature DCs by gently pipetting up and down to ensure all cells are in suspension, then transfer to an appropriate tube.
   NOTE: For phenotype assessment of mature DCs (CD14-CD83+) by flow cytometry, use the following fluorochrome-conjugated antibodies:

   • Anti-Human CD14 Antibody
   • Clone MoP9 (Catalog #60124)
   • Anti-Human CD83 Antibody, Clone HB15e (Catalog #60107)

To minimize non-specific binding, we recommend using Anti-Human CD32 Antibody, Clone IV.3 (Catalog #60012) as an FcR blocker. If labeling mature DCs with Anti-Human CD14 Antibody, Clone M5E2 (Catalog #60004), use Mouse IgG2a, kappa Isotype Control Antibody, Clone MOPC-173 (Catalog #60071) or rat serum in addition to Anti-Human CD32 Antibody, Clone IV.3.