Human MO-DC Differentiation Cytokine Kit

Note: S Size some components will remain.

Preparation of Human Peripheral Blood Monocyte-Derived Dendritic Cells

1. CD14⁺ Monocyte Isolation

1.1 Thaw cryopreserved PBMCs and centrifuge at 300 × g for 5 minutes at room temperature. Discard the supernatant and resuspend the cells in complete RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin-streptomycin). Perform cell counting and viability assessment (viability should be >95%). Isolate CD14⁺ cells using CD14 positive selection magnetic beads (CD14 Nanobeads, human, Cat. No. S0K0004) according to the manufacturer's protocol. After isolation, assess the purity of the CD14⁺ cell population by flow cytometry. To ensure the quality of subsequent experiments, the purity should be >95%.

2. Induction of Monocytes into Immature Dendritic Cells

2.1 Adjust the concentration of the isolated CD14⁺ cells to 1–2 × 10⁶ cells/mL using complete RPMI 1640 medium (with 10% FBS and 1% penicillin-streptomycin). Seed 1 mL per well into a 24-well plate and immediately add the recombinant human cytokines IL-4 (50 ng/mL) and GM-CSF (100 ng/mL). Place the plate in a 37°C incubator with 5% CO₂ to initiate culture.

2.2 After 48 hours of culture, observe the cell morphology under a microscope. If the cells adhere well: gently aspirate half of the old medium and slowly add an equal volume of fresh complete medium containing the same concentrations of IL-4 and GM-CSF. If the cells adhere poorly or are largely in suspension: do not remove the old medium; simply supplement with an appropriate amount of fresh complete medium containing the same concentrations of cytokines.

Continue the culture: After the medium change/supplementation, continue culturing the cells for an additional 72 hours. At the end of this period, immature dendritic cells (iDCs) will be obtained.

3. Maturation of Immature Dendritic Cells into Mature Dendritic Cells

3.1 Gently pipette to harvest the iDCs from step 2 and centrifuge at 300 × g for 10 minutes. Discard the supernatant. Resuspend the cells in fresh complete medium and adjust the density to 5 × 10⁵ cells/mL. Seed the cell suspension into a new 24-well plate and add the maturation inducer cocktail: TNF-α (10 ng/mL), IL-1β (10 ng/mL), IL-6 (50 ng/mL), and PGE₂ (1 μg/mL). Incubate the cells at 37°C in a 5% CO₂ incubator for an additional 48 hours. Upon completion, mature dendritic cells (mDCs) will be obtained and can be used for subsequent experiments.

Flow Cytometry Detection

1. Cell Collection: After cytokine treatment, collect the cells.
2. Cell Counting: Count the cells and calculate the total number. Centrifuge at 300 × g for 5 minutes, discard the supernatant, and resuspend the cells in cell staining buffer (PBS with 10% FBS or 1% BSA) at a concentration of 5–10 × 10⁶ cells/mL.
3. Fc Receptor Blocking: On ice, incubate 100 μL of cell suspension (containing 5–10 × 10⁵ cells) with 1 μL of Human FcR Blocking Reagent (human antibodies against CD16/32/64, Starter Cat: S0F0012) for 30 minutes. Centrifuge at 300 × g for 5 minutes and discard the supernatant.
4. Antibody Staining: Resuspend the cells in 100 μL of PBS with 10% FBS (or 1% BSA). Add the following antibodies for single-tube staining:
   • APC Rabbit Anti-Human CD80 Antibody (Starter)
   • APC Rabbit Anti-Human CD86 Antibody (Starter Cat: S0B1658)
   • Anti-Hu CD83 APC
   • Brilliant Violet 711™ anti-human CD11c Antibody
   • Brilliant Violet 421™ anti-human CD14 Antibody
   • Alexa Fluor® 488 anti-human HLA-DR Antibody
     Incubate at 4°C for 30 minutes. Centrifuge at 300 × g for 5 minutes and discard the supernatant.
5. Cell Washing: Wash the cells with PBS to remove residual antibodies, then resuspend again in PBS.
6. Viability Dye Staining: Add 7-AAD to each tube and incubate at room temperature in the dark for 5 minutes.
7. Perform Flow Cytometry Analysis.

Store in separate containers to minimize freeze-thaw cycles.