Application protocol

TH1, TH2, and TH17 polarization of naive 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 naive CD4+ T cells and in vitro activation and differentiation, through to comprehensive cell analysis. It is demonstrated 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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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 (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

  • MACS SmartStrainers (30 μm) (# 130-098-458)
  • 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)

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 naive CD4+ T cells
 

  • CD45 Antibody, anti-mouse, VioGreen, REAfinity 
  • CD4 Antibody, anti-mouse, VioBlue, REAfinity 
  • CD62L Antibody, anti-mouse, PE, REAfinity 
  • CD3ε Antibody, anti-mouse, APC-Vio 770, REAfinity 
  • CD44 Antibody, anti-mouse, FITC, REAfinity CD44 Antibody, anti-mouse, FITC, REAfinity 

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 plate.

 

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 naive 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 Antibody, anti-human/mouse, PE, REAfinity 
  • Gata3 Antibody, anti-human/mouse, APC, REAfinity 
  • RORγ (t) Antibody, anti-human/mouse, APC, REAfinity
  • IFN-γ, Antibody, anti-mouse, APC, REAfinity 
  • IL-4, Antibody, anti-mouse, PE, REAfinity 
  • IL-17A, Antibody, anti-mouse, PE, REAfinity 
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