CellXVivo Mouse Treg Cell Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # CDK007
Key Product Details
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
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)
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
Coat wells of a 24-well plate with Hamster Anti-Mouse CD3 Antibody.
![Coat wells of a 24-well plate](https://resources.rndsystems.com/images/site/cdk001_st1.png)
Prepare a single cell suspension of mouse splenocytes.
![Prepare a single cell suspension](https://resources.rndsystems.com/images/site/cdk007-step-2.png)
Isolate human naïve CD4+ T cells from PBMCs (e.g., using magnetic cell selection).
![Isolate T Cells from PBMCs](https://resources.rndsystems.com/images/site/cdk005-st2.png)
Perform a cell count.
![Perform a cell count](https://resources.rndsystems.com/images/site/cdk001-st3.png)
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.
![Suspend cells in media](https://resources.rndsystems.com/images/site/cdk001-st4.png)
Verify Treg cell differentiation by analyzing marker expression using flow cytometry.
Mature Treg cells are now ready for further downstream applications.
![Verify Treg cell differentiation](https://resources.rndsystems.com/images/site/cdk001_st5.png)