CellXVivo Human Treg Cell Differentiation Kit

For the differentiation of Human Regulatory T (Treg) Cells from a preparation of naïve CD4+ T cells.

Why Differentiate Treg Cells In Vitro?

Forkhead Box P3 (FoxP3)⁺ regulatory T (Treg) cells are a suppressive subset of CD4⁺ T cells that function to antagonize immune responses. Treg cells have the capacity to prevent potentially damaging autoimmune and protective immune responses, so the number of Treg cells is a crucial determinant of the regulatory burden on the immune system. In vitro differentiation of Treg cells from the larger naïve CD4⁺ T cell population provides increased numbers of Treg cells to facilitate downstream research.

This kit contains the following optimized proteins and reagents to drive efficient differentiation of naïve CD4⁺ T cells into FoxP3⁺CD25⁺ Treg cells.

• Mouse Anti-Human CD3 Antibody
• Human Treg Reagent 1
• Human Treg Reagent 2
• Human Treg Reagent 3
• Human Treg Reagent 4
• Reconstitution Buffer 1
• Reconstitution Buffer 2
• Wash Buffer (20X)

Provides sufficient reagents for the differentiation of one 24-well plate.

Stability and Storage

Store the unopened kit at 2-8 ^°C. After opening the kit, the Mouse Anti-Human CD3 Antibody and Human Treg Reagents 1-4 may be stored at 2-8 ^°C under sterile conditions for up to 30 days or at -20 ^°C to -70 ^°C in a manual defrost freezer for up to 3 months. Reconstitution Buffer 1, Reconstitution Buffer 2, and 20X Wash Buffer may be stored under sterile conditions for up to 3 months at 2-8 ^°C. Storage conditions are valid if used within the expiration date of the kit.

Scientific Data Examples for CellXVivo Human Treg Cell Differentiation Kit

Differentiation of Human CD4+ T Cells into Treg Cells Confirmed by FoxP3 and CD25 Expression. Human peripheral blood naïve CD4+T cells were incubated with reagents included in the Human Treg Cell Differentiation Kit for 5 days. Cells were fixed, permeabilized, and stained using the FlowX™Human Regulatory T Cell Kit (Catalog # FMC021). Cells were imaged using flow cytometry. Quadrants were set based on isotope-stained samples.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, mature human CD4+ T cells can be differentiated into Tregs using the following procedure:

• Coat a plate with Mouse Anti-Human CD3 Antibody
• Isolate naïve CD4⁺ T cells from Human PBMCs
• Culture naïve CD4⁺ T cells in Human Treg Differentiation Media for 5 days
• Verify differentiation into Treg cells by flow cytometry

View Graphic Procedure

 

 

Reagents Provided

Reagents Supplied in the CellXVivo™ Human T (Treg) Cell Differentiation Kit (Catalog # CDK006):

• Mouse Anti-Human CD3 Antibody
• Human Treg Reagent 1
• Human Treg Reagent 2
• Human Treg Reagent 3
• Human Treg Reagent 4
• Reconstitution Buffer 1
• Reconstitution Buffer 2
• Wash Buffer (20X)

 

Other Supplies Required

Reagents

• Ficoll-Hypaque™
• MagCellect™ Human Naïve CD4⁺ T Cell Isolation Kit (R&D Systems, Catalog # MAGH115, or equivalent)
• X-VIVOTM15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza, or equivalent)

Equipment

• Tissue culture flasks and/or plates
• Sterile deionized water
• Microscope
• Hemocytometer
• 37 °C and 5% CO₂ incubator
• Centrifuge
• Pipettes and pipette tips

 

Procedure Overview

Coat wells of a 24-well plate with Mouse Anti-Human CD3 Antibody.

Isolate PBMCs from human blood.

Isolate human naïve CD4⁺ T cells from PBMCs (e.g., using magnetic cell selection).

Perform a cell count.

Suspend 1 - 2 x 10⁵ naïve CD4⁺ T cells/mL in Human Treg Differentiation Media.

Culture the cells on plates pre-coated with CD3 antibody for 5 days.

Verify Treg cell differentiation by analyzing marker expression using flow cytometry.

Mature Treg cells are now ready for further downstream applications.