Application protocol

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

In vitro human regulatory T cell expansion

This application protocol describes use of the Treg Expansion Kit, human, which is designed to efficiently expand Treg cells that maintain FoxP3 expression after isolation with the CD4+CD25+ Regulatory T Cell Isolation Kit, human. It includes a complete protocol for your Treg cell workflow, describing human Treg cell isolation from peripheral blood mononuclear cells (PBMCs), their in vitro expansion,  and subsequent flow cytometry analysis. 

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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.

For Treg cell isolation from PBMCs

Treg cell isolation kit

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

Separation buffer

  • 1× PBS (pH 7.2), BSA, EDTA
    or
  • 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 Treg cell in vitro expansion

Expansion medium

  • TexMACS™ Medium, research grade (500 mL), (# 130-097-196)
  • Human IL-2 IS, premium grade (50 µg) (# 130-097-745)
  • human AB Serum
  • β-Mercaptoethanol (β-ME)
  • Penicillin/streptamycin (PenStrep)
  • (Optional) Rapamycin 

Expansion process

  • Treg Expansion Kit, human: 2 mL for stimulation of 1×107 Treg cells (# 130-095-345);
    2×2 mL for stimulation of 2×107 Treg cells (# 130-095-353)
  • 96-well round-bottom plate
  • (Optional) 24-well plate 

(Optional) For bead removal and restimulation

  • MACSiMAG™ Separator (# 130-092-168) 

For surface and intracellular flow cytometry staining of Treg cells

  • Treg Detection Kit (CD4/CD25/FoxP3) (PE), human (# 130-094-163)
    or
  • Treg Detection Kit (CD4/CD25/FoxP3) (APC), human (# 130-094-158)
  • (Optional): CD127- PE-Vio® 770 (# 130-109-516) or CD127- PE-Vio 615, human
    (# 130-107-513)
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