TH cell differentiation | CD4 T cell differentiation | Miltenyi

This complete workflow allows for a reliable and efficient T helper cell (T_H cell) differentiation. Starting with cell isolation of naive CD4⁺ T cells over to in vitro activation and differentiation, up to multiparameter flow cell analysis.

We provide 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 T_H1, T_H2, and T_H17 cells.

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.

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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.

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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 Neumann^1,2, Alexander Scheffold², Nicole Pietz³, Anna Foerster-Marniok³, Michaela Niemöller³, Julia Milleck³, Anne Richter³, and Tobias Schenk³
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

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

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.

Expansion of human CD4⁺and CD8⁺ T cells with T Cell TransAct

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

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 T_H cell polarization using CytoBoxes (T_H1, T_H2, or T_H17) in combination with either RPMI or TexMACS Medium

T_H cell polarization with CytoBoxes

Convenient all-in-one starter kits for polarization of mouse T_H1, T_H2 and T_H17 cells
Recombinant cytokines and functional-grade blocking antibodies

Principle of the Cytokine Secretion Assays

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.

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 T_H cell subsets and corresponding polarizing cytokines, surface markers, transcription factors, and secreted cytokines.

Human T_H cell subsets and corresponding polarizing cytokines, surface markers, transcription factors, and secreted cytokines.

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.