CellXVivo Human Monocyte-derived DC Differentiation Kit

For the differentiation of CD14+ monocytes into dendritic cells
Catalog # Availability Size / Price Qty
CDK004
Morphology of Immature Human Dendritic Cells Cultured in Differentiation Media for 7 days.
4 Images
Product Details
Procedure
Citations (2)
FAQs
Reviews

CellXVivo Human Monocyte-derived DC Differentiation Kit Summary

Kit Summary

For the differentiation of CD14+ monocytes into dendritic cells.

Key Benefits

  • Provides optimized reagents needed to induce immature and mature dendritic cell differentiation from CD14+ monocytes
  • Utilizes validated and straightforward procedures
  • Does not require specialized instrumentation

 

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.

 

Kit Contents

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.

 

Data Examples
Morphology of Immature Human Dendritic Cells Cultured in Differentiation Media for 7 days
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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.

Mature Dendritic Cells Induce Proliferation of Allogeneic T-cells
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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 of 3H-TdR was measured using a scintillation counter. Results are presented as the mean cpm of triplicates.

Phenotypic Analysis of Cultured Immature and Mature Monocyte-derived Dendritic Cells
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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.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

Data Example

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.

CellXVivo Human Monocyte-derived Dendritic Cell Differentiation Kit

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
 

 

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.

Isolate PBMCs from human blood

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

Enrich CD14<sup>+</sup>Cells from PBMCs

Perform a cell count.

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.

Suspend cells in media

Verify immature dendritic cell differentiation by flow cytometry.

The Th2 cells are now ready for further downstream applications.

Verify immature dendritic cell differentiation

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

Induce dendritic cell maturation

Verify dendritic cell maturation by flow cytometry.

Mature dendritic cells are ready for downstream applications.

Verify dendritic cell maturation

Reagents Provided

Citations for CellXVivo Human Monocyte-derived DC Differentiation Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

2 Citations: Showing 1 - 2
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  1. Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells In Vitro
    Authors: HR Freyberger, Y He, AL Roth, MP Nikolich, AA Filippov
    Viruses, 2018;10(11):.  2018
  2. Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model.
    Authors: Allen A, Wang C, Caproni L, Sugiyarto G, Harden E, Douglas L, Duriez P, Karbowniczek K, Extance J, Rothwell P, Orefo I, Tite J, Stevenson F, Ottensmeier C, Savelyeva N
    Cancer Immunol Immunother, 0;0(0):.  0

FAQs

  1. What method should be used for dissociation of cells in CellXVivo Human Monocyte-derived DC Differentiation Kit, Catalog # CDK004? 

    • Cell scraping is prefered but dissociation in 2 mM EDTA can be used as an alternative method. 

  2.  For CellXVivo Human Monocyte-derived DC Differentiation Kit, Catalog #  CDK004, are the dendritic cells harvested on day 7 and day 10 adherent or in suspension?

    • When using Catalog # CDK004, the dendritic cells harvested on day 7 and day 10 are a mixture of  cells in suspension and adherent cells. Typically  ~50-90% of the cells are in  suspension at day 7 and ~40-80% of the cells are suspension at day 10.

  3. The Cloudz NK Cell Expansion protocol, Catalog # CDK004, recommends using T25 flasks. Do you have a protocol or recommended starting cell numbers for larger flasks, such as T75?

    • It is important to maintain a higher density of cells in the beginning of the culture, so start in a T25 and then scale up to a T75/T175 after 6-9 days when the cells are expanding exponentially. The cells expand dramatically at some point between day 6-9, dependent on donor. 

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