CellXVivo Human Treg Cell Differentiation Kit

For the differentiation of human regulatory T (Treg) cells from a preparation of naïve CD4+ T cells
Catalog # Availability Size / Price Qty
CDK006
CellXVivo Human Treg Cell Differentiation Kit
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Product Details
Procedure
Citations (1)
FAQs
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CellXVivo Human Treg Cell Differentiation Kit Summary

Kit Summary

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

Key Benefits

  • Contains high quality bioactive proteins
  • Yields a highly enriched population of FoxP3+CD25+ Treg cells
  • Provides optimized reagents for inducing Tregs from CD4+ T cells
  • Involves validated and straightforward procedures
  • Does not require specialized instrumentation
 

 

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.

 

Kit Contents

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 two 24-well plates.

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.

 

Data Examples
Differentiation of Human CD4+ T Cells
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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.

Specifications

Shipping Conditions
The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Human

Product Datasheets

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
 

 

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.

Coat wells of a 24-well plate

Isolate PBMCs from human blood.

Isolate PBMCs from human blood

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

Isolate T Cells from PBMCs

Perform a cell count.

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.

Suspend cells in media

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

Citation for CellXVivo Human Treg Cell Differentiation Kit

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citation: Showing 1 - 1

  1. Cell-Penetrating Anti-Protein Kinase C Theta Antibodies Act Intracellularly to Generate Stable, Highly Suppressive Regulatory T Cells
    Authors: EI Ozay, S Shanthalin, HL Sherman, JA Torres, BA Osborne, GN Tew, LM Minter
    Mol. Ther., 2020;0(0):.  2020

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