CellXVivo Mouse Treg Cell Differentiation Kit

For the differentiation of regulatory T Cells (Tregs) 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.

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

Stability and Storage

Store the unopened kit at -20 °C. After opening the kit, the Human Anti-Mouse CD3 Antibody, Mouse Treg Reagent 1, and Mouse Treg Reagent 2 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. Mouse Treg Reagent 3 may be stored 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 to 8 °C. Storage conditions are valid if used within the expiration date of the kit.

Scientific Data Examples for CellXVivo Mouse Treg Cell Differentiation Kit

Differentiation of CD4+ T Cells into Treg Cells Confirmed by FoxP3 and CD25 Expression.  Naïve CD4+T cells were incubated without (A, B) or with (C, D) reagents included in the CellXVivo™ Mouse Treg Cell Differentiation Kit for 5 days. Cells were fixed, permeabilized, and stained using the FlowX Mouse Regulatory T Cell Kit (Catalog # FMC022). 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, naïve mouse CD4+ T cells can be differentiated into Tregs using the following procedure:

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

View Graphic Procedure

 

 

Reagents Provided

Reagents Supplied in the CellXVivo™ Mouse Treg Cell Differentiation Kit (Catalog # CDK007):

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

 

Other Supplies Required

Reagents

• MagCellect™ Mouse Naïve CD4⁺ T Cell Isolation Kit (R&D Systems, Catalog # MAGH205, or equivalent).
• RPMI 1640
• Fetal Bovine Serum (FBS)
• β-Mercaptoethanol (2-ME)
• L-Glutamine–Penicillin–Streptomycin solution

Equipment

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

 

Procedure Overview

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

Prepare a single cell suspension of mouse splenocytes.

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

Perform a cell count.

Suspend 0.5 - 1 x 10⁶ naïve CD4⁺ T cells/mL in Mouse 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.