Application protocol

TH1, TH2, and TH17 polarization of naïve CD4+ mouse T cells

A complete workflow for cell preparation, isolation, polarization, and analysis

This application protocol describes a complete workflow for reliable and efficient mouse TH cell differentiation, starting with single-cell preparation, followed by isolation of naïve CD4+ T cells and in vitro activation and differentiation, through to comprehensive cell analysis. We demonstrate that in vitro TH cell differentiation in the presence of TexMACS™ Medium leads to a higher expression level of the characteristic effector cytokines in the various TH subsets compared to RPMI 1640.

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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

Buffer (standard wash and dilution buffer)

  • autoMACS® Rinsing Solution (# 130-091- 222)
  • MACS® BSA Stock Solution (# 130-091-376)

For preparation of a single-cell suspension from mouse spleen

  • Pre-Separation Filters, 30 μm (# 130-041-407)
  • MACSmix™ Tube Rotator (# 130-090-753) 
  • CO2 incubator at 37 °C
  • gentleMACS™  Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334) 
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes

For isolation of naïve CD4+ T cells

  • Naive CD4+ T Cell Isolation Kit, mouse (# 130-104-453)
  • LS Columns (# 130-042-401)
  • MACS Separator for LS columns (e.g., MidiMACS™ Separator # 130-042-302)
  • MACS Multistand  (#130-042-303)

(Optional) For flow cytometry analysis of freshly isolated naïve CD4+ T cells

  • CD45-VioGreen™ (# 130-102-412)
  • CD4-VioBlue® (# 130-102-456)
  • CD62L-PE (# 130-102-543)
  • CD3ε-APC-Vio®770 (# 130-102-306)
  • CD44-FITC (# 130-102-511)
  • Propidium iodide (#130-093-233)

For TH1, TH2, or TH17 polarization

T cell cultivation

  • TexMACS™ Medium (# 130-097-196)
  • Fetal Bovine Serum (FBS)
  • 100× penicillin/streptomycin stock solution
  • 2-Mercaptoethanol 
  • 96-well plate (e.g., Gas-permeable Culture Plate # 150-000-364)

Note: Depending on the cell number to be cultivated and differentiated per well, 6-, 12-, 24- or 48-well plates might be used. Please refer to the protocol to choose the appropriate cell culture dish.


TH cell polarization

  • TH1 cells: CytoBox TH1, mouse (#130-107-761)
  • TH2 cells: CytoBox TH2, mouse (#130-107-760)
  • TH17 cells: CytoBox TH17, mouse (#130-107-758)

Activation of naïve CD4+ T cells

  •  T cell Activation and Expansion Kit, mouse (# 130-093-627)

For removal of MACSiBead Particles

  • MACSiMAG™ Separator (#130-092-168)

For restimulation of polarized T cells and brefeldin A treatment

  • Ionomycin
  • Phorbol 12-myristate 13-acetate (PMA)
  • Brefeldin A

For staining of lineage-specific transcription factors and cytokines

  • Fox P3 Staining Buffer Set (# 130-093-142)
  • Inside Stain Kit (#130-090-477)
  • T-bet-PE, human and mouse (# 130-098-596)
  • Gata3-APC, human and mouse (#130-100-649)
  • RORg(t)-APC, human and mouse (#130-103-838)
  • IFN-γ-APC, mouse (#130-102-340)
  • IL-4-PE, mouse (#130-102-435)
  • IL-17A-PE, mouse (#130-102-344)
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Protocol


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