YILIMART - CD4 CD25 Reg. T Cell Iso. Kit, mouse

The CD4

⁺

CD25

⁺

Regulatory T Cell Isolation Kit, mouse was developed for the isolation of CD4

⁺

CD25

⁺

regulatory T cells (Treg) from single-cell suspensions of mouse spleen and lymph nodes. The isolation is performed in a fast two-step procedure.

Background information

CD4

⁺

CD25

⁺

immunoregulatory T cells have been shown to actively suppress immune responses against autologous and foreign antigens

in vivo

and

in vitro

. CD25, the IL-2Rα chain, is also expressed on activated CD4

⁺

T cells, CD8

⁺

T cells, dendritic cells, and B cells.

Detailed separation procedure

The isolation is performed in a two-step procedure. First, non-CD4

⁺

are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies against CD8, CD11b, CD45R, CD49b, Ter-119 and Anti-Biotin MicroBeads. The labeled cells are subsequently depleted over a MACS

^®

Column. In the second step, the flow-through fraction of pre-enriched CD4

⁺

T cells is labeled with CD25 MicroBeads for subsequent positive selection of CD4

⁺

CD25

⁺

regulatory T cells (Tregs). Use our optimized protocol to save 30 minutes of handling time.

Downstream applications

Regulatory T cells isolated from mouse lymph nodes with the CD4

⁺

CD25

⁺

Regulatory T Cell Isolation Kit were used in coculture experiments with dendritic cells to study priming of dendritic cells for tolerance induction

in vitro

and after adoptive transfer of primed dendritic cells

in vivo

.

¹

Furthermore, they were isolated and used in various models for adoptive transfer experiments

²

, e.g. in an experimental autoimmune thyroiditis mouse model

³

, and a melanoma mouse model where they prevented tumor immunity

⁴

. In addition,

in vitro

suppression assays were performed with isolated regulatory T cells.

^5,6

Columns

For the first magnetic separation (depletion): LD or autoMACS

^®

Columns. For the second magnetic separation (positive selection): MS or autoMACS Columns.

Figure 1

CD4

⁺

CD25

⁺

regulatory T cells were isolated from mouse spleen cell suspension by using the CD4

⁺

CD25

⁺

Regulatory T Cell Isolation Kit, an LD and two MS Columns, a MidiMACS™ Separator and a MiniMACS™ Separator. The cells were fluorescently stained with CD25-PE and CD4‑FITC (A) or Anti-FoxP3-APC (B) and analyzed by flow cytometry using the MACSQuant

^®

Analyzer. Cell debris and dead cells were excluded from analysis based on scatter signals and propidium iodide fluorescence.

A:	B:
Before separation


CD4 ⁺ CD25 ^- T cells


Isolated CD4 ⁺ CD25 ⁺ regulatory T cells

Figure 2
View details

Figure 2

Working scheme of the CD4
⁺
CD25
⁺
Regulatory T Cell Isolation Kit.

Working scheme of the CD4
⁺
CD25
⁺
Regulatory T Cell Isolation Kit.

Selected references
1. Fallarino, F. et al. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212
2. Schwarz, A. et al. (2004) Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity. J. Immunol. 172: 1036-1043
3. Gangi, E. et al. (2005)
  
  IL-10-producing CD4
  ⁺
  CD25
  ⁺
  regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis.
  
  J. Immunol. 174: 7006-7013
4. Turk, MJ. et al. (2004) Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J. Exp. Med. 200: 771-782
5. Kashiwada, M. et al. (2006)
  
  Downstream of tyrosine kinases-1 and Src homology 2-containing inositol 5′-phosphatase are required for regulation of CD4
  ⁺
  CD25
  ⁺
  T cell development.
  
  J. Immunol. 176: 3958-3965
6. Choileain, N. N. et al. (2006) Enhanced regulatory T cell activity is an element of the host response to injury. J. Immunol. 176: 225-236
Brochures and posters

Regulatory T cell research

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CD4 CD25 Reg. T Cell Iso. Kit, mouse

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Background information

Detailed separation procedure

Downstream applications

Columns

Figure 1

Figure 2

Figure 2

Selected references

Brochures and posters

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