CellXVivo Human Monocyte-derived DC Differentiation Kit Best Seller

For the differentiation of CD14+ monocytes into dendritic cells.

Why Generate Monocyte-derived Dendritic Cells Ex Vivo?

Dendritic cells are a heterogeneous population of functionally homologous immune cells that act as key mediators of the innate and adaptive immune responses. Under homeostatic conditions, dendritic cells are present in large numbers in areas of intense antigen exposure such as the skin, lung, and intestine and can be difficult to isolate and harvest. However, in response to an immune stimulus, CD14⁺ monocytes, which are present in high numbers in the periphery and circulation, can differentiate into inflammatory/monocyte-derived dendritic cells. Harvesting CD14⁺ monocytes from peripheral blood mononuclear cells and driving their differentiation into either immature or mature dendritic cells provides an abundant source of monocyte-derived dendritic cells for downstream studies.

This kit contains the following optimized reagents for the differentiation of monocyte-derived dendritic cells.

• Serum-Free Dendritic Cell Base Media
• Recombinant Human IL-4
• Recombinant Human GM-CSF
• Recombinant Human TNF-alpha
• Reconstitution Buffer 2

Stability and Storage

Store the unopened kit at -20 °C. Do not use past the kit expiration date. Opened or reconstituted Serum-Free Dendritic Cell Base Media, Recombinant Human IL-4, Recombinant Human GM-CSF, or Recombinant Human TNF-alpha should be stored at 2-8 °C under sterile conditions for up to 30 days or at -20 °C to -70 °C in a manual defrost freezer for up to 3 months.* Opened Reconstitution Buffer 2 should be stored at 2-8 °C under sterile conditions for up to 3 months.*

*Provided this is within the expiration date of the kit.

Scientific Data Examples for CellXVivo Human Monocyte-derived DC Differentiation Kit

	Morphology of Immature Human Dendritic Cells Cultured in Differentiation Media for 7 days. Human CD14+monocytes were differentiated into immature human dendritic cells by culturing for 7 days using reagents included in this kit. Immature dendritic cells exhibit characteristic dendritic cell morphology.
	Phenotypic Analysis of Cultured Immature and Mature Monocyte-derived Dendritic Cells. Following culture in complete monocyte-derived differentiation media provided in this kit, day 7 immature dendritic cells (top) and day 10 TNF-alpha treated mature dendritic cells (bottom) were stained with the indicated antibodies for DC-SIGN/CD209 (Catalog # MAB161), B7-1/CD80 (Catalog # MAB140), B7-2/CD86 (Catalog # MAB141), CD83 (Catalog # MAB1774; open histograms), or an appropriate isotype control antibody (filled histograms). Stained cells were analyzed using a Becton Dickinson FACSCalibur™.
	Mature Dendritic Cells Induce Proliferation of Allogeneic T-cells. Serial dilutions of day 10 TNF-alpha treated mature dendritic cells were incubated with allogeneic (blue) or autologous (red) human T cells for 3 days.3H-Thymidine (3H-TdR) was added to the culture for the final 18 hours. Cells were harvested and the incorporation of3H-TdR was measured using a scintillation counter. Results are presented as the mean cpm of triplicates.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mature human monocyte-derived dendritic cells can be generated using the following procedure:

• Isolate human CD14⁺ monocytes from a PBMC preparation
• Culture CD14⁺ monocytes in growth factor-supplemented Serum-Free Dendritic Cell Base Media
• Verify differentiation into immature dendritic cells by flow cytometry
• Induce dendritic cell maturation with TNF-alpha
• Verify dendritic cell maturation via flow cytometry

View Graphic Procedure

 

 

Reagents Provided

Reagents Supplied in the Human Monocyte-derived Dendritic Cell Differentiation Kit (Catalog # CDK004):

• Serum-Free Dendritic Cell Base Media
• Recombinant Human IL-4
• Recombinant Human GM-CSF
• Recombinant Human TNF-alpha
• Reconstitution Buffer 2

 

Other Supplies Required

Reagents

• MagCellect™ Human CD14⁺ Cell Isolation Kit (R&D Systems, Catalog # MAGH105, or equivalent).
• Ficoll-Hypaque™
• Penicillin (optional)
• Streptomycin (optional)

Equipment

• Tissue culture flasks and/or plates
• Inverted microscope
• Hemocytometer
• 37 °C and 5% CO₂ humidified cell culture incubator
• Benchtop centrifuge
• Pipettes and pipette tips

 

Procedure Overview

Isolate PBMCs from human blood.

Enrich human CD14⁺ cells from PBMCs (e.g., using magnetic cell selection).

Perform a cell count.

Suspend 1 x 10⁶ CD14⁺ cells/mL in Differentiation Media.

Culture the cells for 7 days.

Add fresh Diferentiation Media every 2-3 days.

Verify immature dendritic cell differentiation by flow cytometry.

The Th2 cells are now ready for further downstream applications.

Induce dendritic cell maturation with TNF-alpha for 3 days.

Verify dendritic cell maturation by flow cytometry.

Mature dendritic cells are ready for downstream applications.