Comprehensive solutions for your Treg human and mouse research

 

  • Fast and gentle isolation and flow sorting of Treg cells and distinct  Treg subsets
  • Optimized culture, activation, and expansion of Treg cells
  • Phenotyping and functional analysis of Treg cells

Research Treg workflow steps

Sample preparation for Tregs

Tregs from mouse tissue

Tregs represent 1–4% of all lymphocytes in secondary lymphoid organs (such as spleen and lymph node) of wild type mice. Spleen and lymph nodes must be dissociated into a single-cell suspension for many downstream applications. Dissociation can be accomplished with specific Tissue Dissociation Kits (e.g. Spleen Dissociation Kit, mouse) manually or in a fast and convenient way by using the automatic gentleMACS™ Dissociator.

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Analysis of CD4+FoxP3+Treg cells in cell suspensions of lamina propria from mouse colons by flow cytometry using Flowlogic™ software.

A. Representative flow cytometry data showing FoxP3 versus CD4 expression, gated on CD3+ lymphocytes. Dead cells were excluded from the analysis. B. Comparison of numbers of viable CD3+CD4+FoxP3+Treg cells in colonic lamina propria, obtained with the gentleMACS Protocol or the manual method. (n=3 per group)

Isolation and cell sorting of Tregs

Isolation

We offer a broad range of products for the isolation of Treg cells according to your starting material and species; human, mouse, and non-human primate. MACS® MicroBeads and Isolation Kits are the best products for isolation of highly pure and viable distinct Treg subsets.

 

MACS® MicroBead Technology is the most trusted and proven cell isolation technology.

  • Minimal labeling and smallest bead size – preserved characteristics and no cell activation.
  • No cell stress – highest viability and no effects on cell functionality.
  • Free epitopes and no bead aggregates – cells can be directly used in downstream applications.
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Isolation of human CD4+CD25+CD127dim/-Regulatory T Cell using MACS® Technology

CD4+CD25+CD127dim/-Tregs were isolated from human PBMCs by using the CD4+CD25+CD127dim/-Regulatory T Cell Isolation Kit II in combination with the MACS Technology, either manually or automatically using the autoMACS® Pro. The cells were fluorescently stained with CD4-FITC, CD25-APC, and CD127-PE, or CD4-FITC, CD127-PE, and Anti-FoxP3-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. Gating was performed according to CD4 expression.

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Isolation of mouse Treg cells using the CD4+CD25+ Regulatory T Cell Isolation Kit, mouse

Regulatory T CellIsolation Kit, mouse. Treg cells were isolated from mouse splenocytes using the CD4+CD25+ RegulatoryT CellIsolation Kit, mouse. The cells were fluorescently stained with CD4-FITC (A) or Anti-FoxP3-APC (B) and analyzed by flow cytometry (Please note that the cells have already been labeled with CD25-PE during isolation.)

Our MACSxpress® Treg Isolation Kit, human has been developed for the isolation of CD4+CD25+ Treg cells from up to 30 mL of freshly drawn anti-coagulated whole blood. Isolate pure Treg cells in only 30 minutes, without the requirement of density gradient centrifugation. 

Sorting of Tregs with MACSQuant® Tyto® Cell Sorter
 

Tregs are fragile and sensitive to mechanical stress. Therefore, gentle isolation is a critical step for accurate downstream (functional and phenotypical) analyses and applications.

The MACSQuant® Tyto® Cell Sorter is revolutionizing cell sorting. Our patented microchip-based technology opens up new possibilities in basic research and medical applications with high-speed multiparameter cell sorting in the safety of a fully enclosed and sterile cartridge system, the MACSQuant Tyto Cartridge.

The MACSQuant Tyto Cell Sorter gives you the opportunity to sort different subtype of Tregs not only for your research purposes but also for cell and gene therapy application. 
 

Treg cell sorting with MACSQuant® Tyto® Cell Sorter.

  • Gentle to cells: Sort and even resort cells under low pressure without compromising cell viability or functionality.
  • Fast and easy handling: No drop delay or laser alignment needed.
  • Full sterility: Contamination-free within the disposable, fully closed MACSQuant Tyto Cartridge.
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Human Treg sorting by MACSQuant® Tyto® CellSorter. 

Treg cells were sorted using the MACSQuant® Tyto® Cell Sorter. The cells were fluorescently stained with CD4-VG, CD25-APC, CD45RA-FITC,CD127-PE.

REAfinity™ Recombinant Antibodies provide complete multiparameter labeling for your Tregs research Flow cytometry and cell sorting analysis. However, REAlease® Fluorochrome Technology not only supports you with complete multiparameter labeling, but provides unlabeled Tregs after the cell sorting.

Culturing, activation and expansion of Tregs 

Activation, expansion, and stimulation of Treg cells can be very challenging. The combination of TexMACS™ Medium, MACS® Cytokines, and Treg expansion kits (human and mouse) are perfectly suited for in vitro culture and expansion of Treg cells.

TexMACS™ Medium is an optimized serum-free cell culture medium developed for the cultivation and expansion of human and mouse T cells and regulatory T cells.
 

Optimal expansion of your isolated Treg cells using Treg Expansion Kit

  • Based on cell-sized MACSiBead™ Particles loaded with CD3 and CD28 antibodies
  • Optimized to perfection and a careful balance of the stimuli enables reliable Tregs expansion
  • Maintenance of Tregs phenotype and FoxP3 expression in vitro.
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Expansion of Treg cells using the Treg Expansion Kit, human. 

Treg cells were isolated from whole blood using the MACSxpress Treg Isolation Kit, human and expanded in TexMACS Medium using the Treg Expansion Kit, human according to the protocol for 21 days. Treg cells were analyzed for their expression of FoxP3 (as a phenotypic marker) and the growth rate was calculated before (A) and after (B) expansion.

Treg analysis 

Treg suppression assay

The Treg Suppression Inspector has been developed for the in vitro functional characterization of human CD4+CD25+regulatory T cells.

Treg in vitro suppression assay

  • An optimized T cell stimulation reagent for a Treg suppression assay
  • Based on Anti-Biotin MACSiBead™ Particles
  • Analysis of Tregs suppression activity by reduction of responder T cells proliferation
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Treg suppression assay using the Treg Suppression Inspector, human.

Treg and Tresp cells were co-cultured at different ratios in the presence or absence (no stimulation) of the Treg Suppression Inspector. Tresp cell proliferation was assessed by 3H-thymidin incorporation (left) added for 16 hours after 5 days of culture or by a cell tracker dilution (right) of the prior stained responder T cells after 5 days of co-culture.

Flow cytometry analysis

Treg Detection Kits (mouse and human)

The Treg Detection Kits are optimized antibody kits with pre-titrated antibodies that ensure a reliable flow cytometric analysis of Treg cells. Kits are ready-to-use cocktail, which conveniently reduces pipetting steps.

REAfinity™ Recombinant Antibodies

REAfinity™ Antibodies are recombinant antibodies that provide superior lot-to-lot consistency and purity compared to mouse or rat hybridoma-derived, monoclonal antibodies.

Treg characterization with REAfinity™ Antibodies

  • Greater reproducibility: High purity and lot-to-lot consistency with less background
  • Easy handling: Eliminates tedious and costly Fc receptor blocking steps
  • One universal isotype: Convenient and cost-saving
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REAfinity™ Recombinant Antibody model

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Gating strategy for identification of different Treg cells subsets

Human Treg cells were isolated from PBMCs by MACS® using the CD4+CD25+CD127dim/-Regulatory T cell Isolation Kit II, human. Viable CD4 cells were identified using the Viability 405/520 Fixable Dye and CD4-APC-Vio 770. Treg cells were stained with CD25-VioBright FITC, CD127-PE-Vio 770, Anti-FoxP3-APC, Anti-Helios-PE, CD45RA-PE-Vio 615, CD45RO-VioBlue, and Anti-Ki-67-PE. The gating strategy depicted identifies the different Treg populations P1, P2 and P3.

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Scientific poster
Vio® Dyes meet REAfinity™ Antibodies – antibody-fluorochrome conjugates for high-resolution multiparameter flow cytometry analysis

Cesar Evaristo, Marco Vahldieck, Susanne Krauthaeuser, Nicole Jansen, Niklas Wilske, Anne Richter, and Christian Dose, Miltenyi Biotec B.V. & Co. KG Bergisch Gladbach, Germany
 

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