StemXVivo Serum-Free Dendritic Cell Base Media

Catalog # Availability Size / Price Qty
CCM003
Differentiation of CD14+ Monocytes into Dendritic Cells. 
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Product Details
Procedure
Citations (2)
FAQs
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StemXVivo Serum-Free Dendritic Cell Base Media Summary

Kit Summary

Pre-optimized base media for successful dendritic cell culture.

Key Benefits

  • Pre-optimized media for dendritic cell culture
  • High lot-to-lot consistency decreases variation
  • Can be supplemented with user-defined cytokines and growth factors
 

 

Why Use Pre-optimized Reagents for the Production of Dendritic Cells from Monocytes and Subsequent Dendritic Cell Culture?

The efficiency of differentiation of common myeloid progenitors into dendritic cells can vary from one experiment to another depending on the media components, the supplements used to drive differentiation, and the quality of the starting cell population.

Inefficient differentiation represents a costly and time-consuming obstacle that can be minimized through the use of high quality media and differentiation supplements. StemXVivo® Serum-Free Dendritic Cell Base Media is a defined media optimized for dendritic cell culture. The addition of Recombinant Human GM-CSF (Catalog # 215-GM) and Recombinant Human IL-4 (Catalog # 204-IL) generates a defined medium that efficiently drives differentiation of CD14+ monocytes into monocyte-derived dendritic cells (MoDCs).

StemXVivo® Serum-Free Dendritic Cell Base Media:

  • Optimized for dendritic cell culture.
  • High lot-to-lot consistency decreases variation.
  • Can be supplemented with user-defined growth factors and cytokines.
  • Optimized for differentiation of CD14+ monocytes into dendritic cells.

 

Media Components

Reagents supplied in the StemXVivo® Serum-Free Dendritic Cell Base Media (Catalog # CCM003):

250 mL StemXVivo® Serum-Free Dendritic Cell Base Media

Stability and Storage

Upon receipt, the Human StemXVivo® Serum-Free Dendritic Cell Base Media should be stored at <-20 °C in a manual defrost freezer. The media can be thawed at 2 °C to 8 °C or at room temperature. Thawed media can be aliquoted and stored at <-20 °C in a manual defrost freezer for up to 3 months or used within a month when stored in the dark at 2 °C to 8 °C. Avoid repeated freeze-thaw cycles.

Precautions

The human origin-derived components used in this product have been derived from human plasma, which has been tested and found negative for antibodies to HIV-1/2, hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV). However, the medium should be handled as if potentially infectious. Safe laboratory procedures should be followed and protective clothing should be worn when handling this media. The acute and chronic effects of over-exposure to this media are unknown.

Limitations

  • FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
  • The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
  • This reagent should not be used beyond the expiration date indicated on the label.
  • Results may vary due to variations between dendritic cell precursors/progenitor cells derived from different donors.

 

Data Examples
Differentiation of CD14+ Monocytes into Dendritic Cells
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Differentiation of CD14+ Monocytes into Dendritic Cells. LPS-matured monocyte-derived dendritic cells were obtained after CD14+-enriched monocytes were cultured for nine days in StemXVivo® Serum-Free Dendritic Cell Base Media (Catalog # CCM003) supplemented with GM-CSF (Catalog # 215-GM) and IL-4 (Catalog # 204-IL).

Phenotypic Analysis of Cultured Monocyte-derived Dendritic Cells Before and After LPS-induced Maturation
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Phenotypic Analysis of Cultured Monocyte-derived Dendritic Cells Before and After LPS-induced Maturation. Immature monocyte-derived dendritic cells (MoDCs) (open histograms; green line) were obtained after CD14+-enriched monocytes were cultured for seven days in StemXVivo® Serum-Free Dendritic Cell Base Media (Catalog # CCM003) supplemented with 50 ng/mL Recombinant Human GM-CSF (Catalog # 215-GM) and 35 ng/mL Recombinant Human IL-4 (Catalog # 204-IL). Mature monocyte-derived dendritic cells (filled histograms) were cultured under the same conditions for seven days and then induced with LPS for an additional 48 hours. Day seven immature MoDCs and day nine LPS-treated MoDCs were stained with a FITC-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody (Catalog # FAB140F), a FITC-conjugated Mouse Anti-Human CD83 Monoclonal Antibody (Catalog # FAB1774F), a FITC-conjugated Mouse Anti-Human B7-2/CD86 Monoclonal Antibody (Catalog # FAB141F), an Anti-MHC Class II Antibody, or an appropriate isotype control antibody (empty histogram with solid line).

Mature Monocyte-derived Dendritic Cells Induce Proliferation of Allogenic T Cells
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Mature Monocyte-derived Dendritic Cells Induce Proliferation of Allogenic T Cells. CD14+ monocytes were cultured for seven days in Human StemXVivo® Serum-free Dendritic Cell Base Media (Catalog # CCM003) supplemented with Recombinant Human GM-CSF (Catalog # 215-GM), Recombinant Human IL-4 (Catalog # 204-IL) and Gentamycin. The cells were subsequently treated with LPS for an additional 48 hours to induce dendritic cell maturation. Graded doses of mature monocyte-derived dendritic cells were incubated with 1 x 105 autologous or allogenic CD3+ T cells for five days. 3H-thymidine (3H-TdR) was added to the culture for the final 18 hours and T cell proliferation was measured using a scintillation counter. Results are presented as the mean cpm obtained from three experiments.

HSC Differentiation &
Lineage Marker Schematic


Hematopoietic Stem Cells & Lineage-specific Markers

View Interactive Pathway

The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long mainte­nance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continu­ously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage deple­tion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

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

Differentiation of CD14+ Monocytes into Dendritic Cells.  LPS-matured monocyte-derived dendritic cells were obtained after CD14+-enriched monocytes were cultured for nine days in StemXVivo Serum-Free Dendritic Cell Base Media (Catalog # CCM003) supplemented with GM-CSF (Catalog # 215-GM) and IL-4 (Catalog # 204-IL).

Phenotypic Analysis of Cultured Monocyte-derived Dendritic Cells Before and After LPS-induced Maturation.  Immature monocyte-derived dendritic cells (MoDCs) (open histograms; green line) were obtained after CD14+-enriched monocytes were cultured for seven days in StemXVivo Serum-Free Dendritic Cell Base Media (Catalog # CCM003) supplemented with 50 ng/mL Recombinant Human GM-CSF (Catalog # 215-GM) and 35 ng/mL Recombinant Human IL-4 (Catalog # 204-IL). Mature monocyte-derived dendritic cells (filled histograms) were cultured under the same conditions for seven days and then induced with LPS for an additional 48 hours. Day seven immature MoDCs and day nine LPS-treated MoDCs were stained with a FITC-conjugated Mouse Anti-Human B7-1/CD80 Monoclonal Antibody (Catalog # FAB140F), a FITC-conjugated Mouse Anti-Human CD83 Monoclonal Antibody (Catalog # FAB1774F), a FITC-conjugated Mouse Anti-Human B7-2/CD86 Monoclonal Antibody (Catalog # FAB141F), an Anti-MHC Class II Antibody, or an appropriate isotype control antibody (empty histogram with solid line).

Mature Monocyte-derived Dendritic Cells Induce Proliferation of Allogenic T Cells. CD14+monocytes were cultured for seven days in Human StemXVivo Serum-free Dendritic Cell Base Media (Catalog # CCM003) supplemented with Recombinant Human GM-CSF (Catalog # 215-GM), Recombinant Human IL-4 (Catalog # 204-IL) and Gentamycin. The cells were subsequently treated with LPS for an additional 48 hours to induce dendritic Cell Differentiation/Maturation. Graded doses of mature monocyte-derived dendritic cells were incubated with 1 x 105autologous or allogenic CD3+T cells for five days.3H-thymidine (3H-TdR) was added to the culture for the final 18 hours and T cell proliferation was measured using a scintillation counter. Results are presented as the mean cpm obtained from three experiments.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, CD14+ monocytes are differentiated into dendritic cells using the following procedure:

  • Culture CD14+ cells in StemXVivo® Serum-Free Dendritic Cell Base Media supplemented with GM-CSF, IL-4, and Gentamicin.
  • Replace the media every three days.
  • Harvest immature MoDCs or use LPS to induce maturation
 

 

Reagents Provided

Reagents supplied in the StemXVivo® Serum-Free Dendritic Cell Base Media (Catalog # CCM003):

  • 250 mL StemXVivo® Serum-Free Dendritic Cell Base Media

 

Other Supplies Required

Reagents

  • Human CD14+ monocytes isolated using the MagCellect Human CD14+ Cell Isolation Kit (Catalog # MAGH105) or equivalent
  • Recombinant Human GM-CSF (Catalog # 215-GM)
  • Recombinant Human IL-4 (Catalog # 204-IL)
  • LPS
  • Gentamicin (Tocris Catalog # 4131)

Materials

  • Tissue culture flasks (25 cm2 or 75 cm2) or plates (6-well or 24-well)
  • 15 mL centrifuge tubes
  • Serological pipette
  • Pipettes and pipette tips
  • Hemocytometer

Equipment

  • 37 °C and 5% CO2 incubator
  • Centrifuge
  • Vortex mixer
  • Inverted Microscope

 

Procedure Overview

Differentiation of CD14+ Monocytes into Monocyte-derived Dendritic Cells Using StemXVivo® Serum-Free Dendritic Cell Base Media (Catalog # CCM003)

Thaw Human StemXVivo® Serum-Free Dendritic Cell Base Media.

Mix the thawed media by vortexing and warm the media in a 37 °C incubator.

Human StemXVivo Serum-Free Dendritic Cell Base Media

Prepare the CD14+ cells from peripheral blood mononuclear cells.

Resuspend the purified CD14+ cells in Human StemXVivo® Serum-Free Dendritic Cell Base Media.

Resuspend the purified CD14+ cells in Human StemXVivo Serum-Free Dendritic Cell Base Media.

Perform a cell count and adjust the cell density to 1 x 106 cells/mL.

Supplement the media with Gentamicin, GM-CSF, and IL-4.

Add the cell suspension to the tissue culture flask or tissue culture plate.

Perform a cell count

Incubate the cells in a 37 °C and 5% CO2 humidified incubator for 7 days.

Change the media every three days.

Incubate the cells

Observe immature monocyte-derived dendritic cells (MoDCs) from day 5 to day 7.

Induce maturation of MoDCs by adding LPS to the cell suspension on day 7 and incubating for 48 hours.

Add LPS to the cell suspension

Reagents Provided

Citations for StemXVivo Serum-Free Dendritic Cell Base Media

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. CD14-independent responses induced by a synthetic lipid A mimetic.
    Authors: Legat A, Thomas S, Hermand P, Van Mechelen M, Goldman M, De Wit D
    Eur J Immunol, 2010;40(3):797-802.  2010
  2. TLR-stimulated CD34 stem cell-derived human skin-like and monocyte-derived dendritic cells fail to induce Th17 polarization of naive T cells but do stimulate Th1 and Th17 memory responses.
    Authors: Duraisingham SS, Hornig J, Gotch F, Patterson S
    J. Immunol., 2009;183(4):2242-51.  2009

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