| Description |
The CD4+CD62L+ T Cell Isolation Kit II has been developed for the improved isolation of CD4+CD62L+ T helper cells from spleen and lymph nodes. The new kit allows the isolation of naive CD4+ T cells with even better purity and recovery due to refinement of the depletion cocktail, including the addition of a CD25 and an anti-TCRγ/δ+ antibody.
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. 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. |
| 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. |
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| 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. |
| Spleen cells before separation |
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| Pre-enriched CD4+ T cells after depletion of non-CD4+ T cells |
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| Isolated CD4+CD62L+ T cells |
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