Application protocol

Generation of Mo-DCs

This application protocol describes a reliable and reproducible protocol for the generation of monocyte-derived dendritic cells (Mo-DCs). Using high-quality reagents that ensure cell viability, the resulting Mo-DCs can be used for downstream phenotypic and functional characterization.

Protocol

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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step. 

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

General reagent and instrument requirements

  • PBE Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091‑222). Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
    Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not
    recommended for use.
  • Complete medium: Prepare a medium from RPMI 1640 containing 2 mM L-glutamine and 1 % autologous plasma.
  • Phosphate-buffered saline (PBS)
  • Fetal calf serum (FCS)
  • Bovine serum albumin (BSA)
  • RPMI 1640
  • Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells
  • MACSQuant® Analyzer 10 (# 130-096-343) or similar flow cytometry analyzer plus analysis software, such as MACSQuantify™ Software (# 130-094-556)

For isolation of monocytes

  • CD14 MicroBeads, human (# 130-050-201) or CD14 MicroBeads, human – lyophilized (# 130-097-052)
  • Pre-Separation Filters, 30 μm (# 130-041-407) to remove cell clumps.
  • MACS Columns and MACS Separators: CD14+ cells can be enriched using MS, LS, or XS Columns, or positive selection can also be performed using the autoMACS Pro or the autoMACS Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×1072×108

MiniMACS™, OctoMACS™,

VarioMACS, SuperMACS II

LS1×1082×109

MidiMACS™, QuadroMACS™,

VarioMACS, SuperMACS II

XS1×1092×1010SuperMACS II
autoMACS2×1084×109autoMACS Pro, autoMACS
Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
  • (Optional) Fluorochrome-conjugated CD14 antibodies for flow cytometry analysis, e.g., CD14-FITC (# 130-080-701). Learn more about our antibodies and dyes.
  • (Optional) Dead Cell Removal Kit (# 130-090-101) for the depletion of dead cells.

For cell culture, differentiation and maturation of Mo-DCs

  • Human GM-CSF, premium grade (# 130-093-866)
  • Human IL-1β, premium grade (# 130-093-898)
  • Human IL-4, premium grade (# 130-093-922)
  • Human IL-6, premium grade (# 130-093-932)
  • Human TNF-α, premium grade (# 130-094-024)
  • Human PGE2

▲ Note: Learn more about different quality grades and additional package sizes of our cytokines and growth factors.

For flow cytometry analyses of Mo-DCs

  • Anti-HLA-DR-FITC, human
  • Anti-HLA-ABC-FITC, human
  • CD1a-PE, human
  • CD14-FITC, human
  • CD25-APC, human
  • CD40-APC, human
  • CD54-PE, human
  • CD80-PE, human
  • CD83-APC, human
  • CD86-PE, human
  • CD197 (CCR7)-APC, human
  • CD206-APC, human
  • CD209 (DC-SIGN)-APC, human

▲ Note: Learn more about different package sizes and conjugates of our antibodies and dyes.

For antigen uptake capacity assessment

  • FITC-labeled dextran
  • PBS supplemented with 1 % fetal calf serum (FCS)
  • PBS supplemented with 0.5 % BSA

For migratory capacity assessment

  • 24-well Transwell® plates (Corning; pore size 5 µm)
  • RPMI 1640 supplemented with 10 % FCS
  • Human CCL19 (MIP-3β), research grade (# 130-093-969)

For T cell priming capacity assessment

  • Naive CD4T cell Isolation Kit II, human (# 130-094-131)
  • CD4-PE, human
  • CD45RO-APC, human
  • CD45RA-PE, human
  • CellTrace™ Violet solution (Life Technologies®)
  • MLR medium: Prepare a medium from RPMI 1640 containing 2 mM L-glutamine, non-essential amino acids, 0.1 mM sodium pyruvate, 5 % human AB serum.

For cytokine secretion assessment

  • Human CD40-Ligand, premium grade (# 130-096-713)
  • ELISAs for IL12p70 and IL-10 (eBioscience)
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