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CellXVivo Human Monocyte-derived DC Differentiation Kit

For the differentiation of CD14+ monocytes into dendritic cells.

Key Product Details

Morphology of Immature Human Dendritic Cells Cultured in Differentiation Media for 7 days.
(4)
Phenotypic Analysis of Cultured Immature and Mature Monocyte-derived Dendritic Cells.
Mature Dendritic Cells Induce Proliferation of Allogeneic T-cells.
CellXVivo Human Monocyte-derived Dendritic Cell Differentiation Kit
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CDK004

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

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    Verified Customer | Posted 11/09/2020

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FAQs

  • 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. 

  •  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.

  • 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. 

Product Documents

Certificate of Analysis

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