Determine the fate of your T cells

  • Isolation of naïve CD4+ T cells
  • Polarization of  CD4+ T helper cells
  • Detection and enrichment of cytokine-secreting cells
  • Multiparameter flow analysis of T cells

A complete workflow for reliable and efficient T helper cell (TH cell) differentiation, starting with cell isolation of naïve CD4+ T cells and in vitro activation and differentiation, through to comprehensive cell analysis.

Miltenyi Biotec provides gentle cell separation strategies, as well as reliable cell culture reagents such as T cell activation tools, T cell media and polarizing cytokines for the efficient differentiation of TH1, TH2 and TH17 cells.

Application protocols

Discover our T helper cell differentiation workflows

Application data by workflow step

T cell isolation


Isolation of naïve CD4+ T cells

Choose from a variety of MicroBeads and T cell Isolation kits for the separation of human and mouse naïve CD4+ T helper cells.

Naive T Cell Isolation Kits mouse and human

The Naïve T Cell Isolation Kits were developed for the isolation of untouched naïve T cells from human peripheral blood or mouse spleen single cell suspensions. Naïve T cells are ready for downstream applications, e.g., in studies on naive T helper cell activation and differentiation as well as and in functional studies of DCs.

Before separation:
After separation:

Isolation of untouched naive CD4+ T helper cells from PBMCs using the Naive CD4+ T Cell Isolation Kit II, human. Cells were stained with CD4 and CD45RA antibodies and analyzed by flow cytometry using the MACSQuant® Analyzer.

Before separation:
After separation:

Naïve CD4+ T cells were isolated from a single-cell suspension from mouse spleen using the Naive CD4+ T Cell Isolation Kit, mouse. Cells were fluorescently stained with CD45-VioGreen, CD4-VioBlue®, CD62L-PE, CD3ε-APC-Vio770™ , and CD44-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence and a gate was set on CD45+ CD3e+ CD4+ cells.

CD62L, CD45RA and CD45RO MicroBeads 
Combine pre-enrichment with the CD4+ T cell Isolation Kits by positive selection or depletion with the respective MicroBeads to isolate even the rarest naïve CD4+ T cell populations.

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Scientific poster

T helper cells: a complete workflow for cell preparation, isolation, stimulation, polarization and analysis
Christian Neumann1,2, Alexander Scheffold2, Nicole Pietz3, Anna Foerster-Marniok3, Michaela Niemöller3, Julia Milleck3, Anne Richter3, and Tobias Schenk
1German Rheumatism Research Centre Berlin, an Institute of the Leibniz-Association, Berlin, Germany; 2Department of Rheumatology and Clinical Immunology, Charité University Hospital Berlin, Germany; 3Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany


T cell culture

T cell media

TexMACS™ Medium is an optimized, serum-free cell culture medium, developed for reliable T cell and Treg cell culture results. Chemically-defined and without animal-derived components, it allows for the highest reproducibility in human and mouse cell culture applications.

Comparison of expansion rates of human CD4+ and CD8+ T cells in TexMACS Medium or competitor’s T cell medium using the T Cell Activation/Expansion Kit, human
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TexMACS Medium

  • Free of serum and other animal-derived components

  • Reliable T cell activation and high expansion rates

  • Up to MACS® GMP grade

  • Available with or without phenol red

T cell activation

We offer a variety of polyclonal T cell stimulation tools for you to choose from. Find the options below:

 Expansion of human and mouse CD4+ and CD8+ T cells using T Cell Activation/Expansion Kits
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T cell Activation/Expansion Kits 

  • MACSiBead™ Particles loaded with functional-grade antibodies
  • Efficient activation and expansion of human, mouse, and non-human primate T cells or Treg cells.



Activation and expansion of CD4+ and CD8+ T cells with T Cell TransAct
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T Cell TransAct™ Stimulation Reagent

  • Ready-to-use reagent for activation and expansion of human T cells

  • Simple volumetric dosage and easy removal by washing

  • Up to MACS GMP grade

T cell polarization

We provide a broad selection of superior-quality cytokines, with high biological activity at a competitive price. Three different grades, research, premium, and MACS® GMP grade, provide flexibility in your assay setup.

Human IL-2 IS biological activity
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MACS® Premium-Grade Cytokines 

  • Well-defined biological activities, normalized to international reference standards (IU/mg)

  • Exact unit dosing for reproducible results

  • Lot specific activity, requiring no laborious pre-testing
Mouse TH cell polarization using CytoBoxes (TH 1, TH 2, or TH 17) in combination with either RPMI or TexMACS Medium
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TH cell polarization with CytoBoxes 

  • Convenient all-in-one starter kits for polarization of mouse TH1, TH2 and TH17 cells

  • Recombinant cytokines and functional-grade blocking antibodies

Detection and enrichment of cytokine-producing T cells

Cytokine secretion assays are designed for highly sensitive detection and magnetic enrichment of viable cytokine-secreting cells. This unique technology enables detection of one antigen-specific T cell per million cells. Isolated cells can be immediately used for downstream applications, e.g., cell culture and flow cytometric analysis.

A range of kits are available for human and mouse cells secreting TNF-α, GMCSF, IFN-α, IFN-ƴ, or IL-2, -4, -5, -10, -12, -13, -17, -22.

Principle of the MACS Cytokine Secretion Assays
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Cytokine Secretion Assay –  Detection Kits 

  • Accurate analysis of cytokine secretion at single-cell level

  • Sensitive detection of one T cell per million cells

Cytokine Secretion Assay – Cell Enrichment and Detection Kits

  • Unique technology for the isolation of viable cytokine-secreting cells

  • Additional magnetic enrichment step


Characterization of CD4+ T cells

Mouse TH cell subsets and corresponding polarizing cytokines, surface markers, transcription factors, and secreted cytokines.
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Human TH cell subsets and corresponding polarizing cytokines, surface markers, transcription factors, and secreted cytokines.
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Multiparameter flow analysis of T cells

As the final step in most workflows, flow analysis plays an important part in various T cell applications. Our flow reagents can help you ensure that the data you generate on your flow cytometer is reliable and consistent:

  • REAfinity™ Recombinant Antibodies – recombinantly generated antibodies that ensure lot-to-lot consistency and eliminate background signal
  • MACSplex™ Cytokine Kits – analysis of multiple soluble cytokines in one sample using any standard flow cytometer
  • Rapid Cytokine Inspector – combined surface marker and intracellular cytokine staining for multiparameter analysis of activated human T cells
  • Cytokine Secretion Assay – Detection Kits – flow analysis of cytokine secretion at a detection level of one T cell in a million

Build your own T cell flow panel with our Flow Panel Builder

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Application note
Flow cytometry analysis of TH subsets

Find out more about multiparameter flow analysis of your favorite TH cell subsets.

Related information


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