Application protocol

Human CD4+CD25+ regulatory T cell isolation, in vitro suppression assay, and analysis

In vitro human regulatory T cell suppression assay

This application protocol describes every step from human Treg and Tresp cell isolation, to their co-cultivation in an in vitro suppression assay using the Treg Suppression Inspector, and flow cytometry analysis. 

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


The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For isolation of CD4+CD25+ Treg cells

Treg cell isolation kit

  • CD4+CD25+ Regulatory T Cell Isolation Kit, human (# 130-091-301) 

Separation buffer

  • 1× PBS (pH 7.2), BSA, EDTA
  • 6× 1.5 L autoMACS® Running Buffer – MACS® Separation Buffer (# 130-091-221)  

Magnetic separation

  • LD Columns, 1 for 1×108 labeled cells (# 130-042-901)
  • MS Columns, 2× per isolation (# 130-042-201)
  • MidiMACS™ Separator (Separator for LD Column), (# 130-042-302)
  • MiniMACS™ Separator (Separator for MS Column), (# 130-042-102)
  • MACS MultiStand (# 130-042-303)
  • (Optional) Pre-Separation Filters, 30 µm (# 130-041-407) 

For surface immunofluorescent staining of Treg and Tresp cells and flow cytometry analysis

Miltenyi Biotec provides flow cytometry antibodies coupled to a variety of different fluorochromes. We recommend using the antibodies listed below. 
  • CD4-VioBlue, Clone: VIT4, 100 tests # 130-094-153
  • Clone: M-T466, 100 tests # 130-097-333
  • CD25 Antibody, anti-human, PE, REAfinity™ # 130-115-534

For in vitro suppression assay

Suppression medium

  • TexMACS™ Medium, research grade (500 mL), (# 130-097-196)
  • Human AB Serum
  • Penicillin/streptamycin (PenStrep) 

Suppression assay

  • Treg Suppression Inspector, human (# 130-092-909), for stimulation of 5×107 total cells
  • 96-well flat-bottom plate
  • CellTrace™ Violet Cell Proliferation Kit, for flow cytometry 
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