CD4<SUP>+</SUP>CD62L<SUP>+</SUP> T Cell Isolation Kit II

CD4⁺CD62L⁺ T Cell Isolation Kit II

Unrivaled: excellent purity and unmatched recovery
Time-saving: cells can be immediately used for downstream applications
Reliable: proven in a plethora of publications

Description

The CD4⁺CD62L⁺ T Cell Isolation Kit II was developed for the isolation of mouse CD4⁺CD62L⁺ T helper cells from spleen and lymph nodes. The isolation is performed in a two-step procedure.

Details

Background information
CD62L (L-selectin) is highly expressed on naive T cells and down-regulated upon activation. It is also expressed on a small subset of memory T helper cells, the central memory T cells, which can be distinguished from naive T helper cells by their high expression of CD44. Furthermore, CD62L is expressed on most thymocytes, naive CD8⁺ T cells, B cells, dendritic cells, macrophages, NK cells, neutrophils, eosinophils, regulatory T cells, and TCRγ/δ⁺T cells.

Detailed separation procedure
For isolation of CD4⁺CD62L⁺ T helper cells, the non-T helper cells as well as regulatory T cells and γ/δ T cells are depleted by indirect magnetic labeling using a cocktail of lineage-specific biotin-conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), and Ter-119, as well as antibodies against CD25 and TCRγ/δ in combination with Anti-Biotin MicroBeads. Subsequently, CD4⁺CD62L⁺ T cells are positively selected from the enriched CD4⁺ T helper cell fraction with CD62L (L-selectin) MicroBeads.

Downstream applications
CD4⁺CD62L⁺ T helper cells isolated with the CD4⁺CD62L⁺ T Cell Isolation Kit II can be used for the analysis of T cell activation by antigen-presenting cells, for studies on cytokine expression and receptor signaling, or for adoptive transfer experiments. Naive T cells, isolated with the CD4⁺CD62L⁺ T Cell Isolation Kit, have been used for in vitro polarization into Th1 and Th2 cells and were further analyzed for cytokine production after treatment with IL-33.¹ In addition, isolated naive T cells were used in a study about the influence of the golli protein (an alternatively spliced product of the myelin basic protein gene) on the regulation of calcium influx in T cells. Isolated naive T cells were e.g. analyzed by calcium imaging as well as in patch clamp experiments.²

Columns

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

Figure 1

CD62L⁺ T cells were isolated from a mouse spleen cell suspension using the CD4⁺ CD62L⁺ T Cell Isolation Kit, an LS and an MS Column, a MidiMACS™ and a MiniMACS™Separator. The cells were fluorescently stained with CD4-FITC and CD62L-APC for detection of naive T cells and with CD62L-APC and CD44-PE for detection of central memory T cells. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

Isolated CD4⁺CD62L⁺ T cells

CD62L<sup>+</sup> T cells were isolated from a mouse spleen cell suspension using the CD4<sup>+</sup> CD62L<sup>+</sup> T Cell Isolation Kit, an <a href="/en/PG_115_163_LS_Columns.aspx">LS</a> and an <a href="/en/PG_115_162_MS_Columns.aspx">MS Column</a>, a <a href="/en/PG_124_182_MidiMACS_Separator.aspx">MidiMACS™</a> and a <a href="/en/PG_124_180_MiniMACS_Separator.aspx">MiniMACS™Separator</a>. The cells were fluorescently stained with <a href="/en/PG_595_212_CD4.aspx">CD4-FITC</a> and <a href="/en/PG_595_322_CD62L.aspx">CD62L-APC</a> for detection of naive T cells and with CD62L-APC and CD44-PE for detection of central memory T cells. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

Spleen cells before separation

Pre-enriched CD4⁺ T cells after depletion of non-CD4⁺ T cells

CD4+CD62L+ T Cell Isolation Kit II

CD4⁺CD62L⁺ T Cell Isolation Kit II