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At a glance: Kits and reagents for the separation of Treg cells from spleen and lymph nodes
|Starting material||Isolation strategy||Comments||Automation||Product|
|Isolation of Treg cells|
|Single-cell suspension from spleen/lymph node||Combination of depletion of non-target cells and subsequent positive selection of target cells||Final positive (Treg cells) and negative (T cells) fractions can both be used for downstream assays/analysis||Yes*||CD4+CD25+ Regulatory T Cell Isolation Kit, mouse|
|Pre-enrichment of Treg cells|
|Single-cell suspension from spleen/lymph node||Positive selection of target cells||Isolation of CD25+ cells||Yes*||CD25 MicroBead Kit, mouse|
|Single-cell suspension from spleen/ lymph node||Positive selection of target cells||Isolation of all CD4+ cells||Yes*||CD4 (L3T4) MicroBeads, mouse|
|Single-cell suspension from spleen/ lymph node||Isolation via depletion of non-target cells||Depletion of all CD8+ and non-T cells.||Yes*|
|*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X.|
Treg cells can be isolated easily and fast from spleen and lymph node cell suspensions by using the Regulatory T Cell Isolation Kit, mouse.
Treg cells from FoxP3 reporter mice, where FoxP3-positive cells co-express a fluorescent protein like GFP or RFP, are usually isolated by flow cytometry-based sorting. However, magnetic enrichment prior to flow sorting is useful to increase the target cell frequency. For this purpose, CD4 MicroBeads, CD4+ T Cell Isolation Kit, and CD25 MicroBead Kit are the best choice.
Mouse Treg cells can be distinguished from other T cells based on the surface markers CD4 and CD25 and more specifically by the transcription factor FoxP3. As there is no unique marker available for Treg cell identification, it is crucial to have a specific marker panel to avoid interference from other cells during flow cytometry analysis.
Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ antibodies and flow analysis kits for comprehensive analysis.
At a glance: Kits and reagents for the analysis of cytokines and transcription factors of Treg cells by flow cytometry
|Intracellular staining of FoxP3||Buffer set optimized to be used with Anti-FoxP3 antibodies||FoxP3 Staining Buffer Set|
|Detection of Treg cells||Treg Detection Kit (CD4/CD25/FoxP3), mouse|
|Detection of IL-10 secretion||Enables cell enumeration||Mouse IL-10 Secretion Assay – Detection Kits|
|Detection and enrichment of IL-10 secreting cells||Mouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE)|
Since the transcription factor FoxP3 is one of the most reliable markers to identify Treg cells, intracellular staining is an important technique for flow cytometry analysis. Miltenyi Biotec offers a ready-to-use FoxP3 Staining Buffer Set which has been specifically developed to ensure effective staining with the various fluorochrome-conjugated Anti-FoxP3 antibodies.
Dedicated mouse Treg Detection Kits (CD4/CD25/FoxP3) (PE) or (APC), which include fluorochrome-conjugated CD4, CD25, and Anti-FoxP3 antibodies and the FoxP3 Staining Buffer Set, allow the detection of Treg cells based on both surface (CD4 and CD25) and intracellular markers (FoxP3).
Under certain conditions, Treg cells secrete anti-inflammatory cytokines such as IL-10. These IL-10–secreting Treg cells can be detected at a single-cell level with the Mouse IL-10 Secretion Assay – Detection Kits (PE) or (APC). In addition, IL-10–secreting cells can also be enriched by using the Mouse IL-10 Secretion Assay – Cell Enrichment and Detection Kit (PE).
At a glance: Kits and reagents for the expansion of Treg cells
|Dedicated expansion kit||Provides optimized stimulation conditions for in vitro Treg cell expansion||Treg Expansion Kit, mouse|
|Supplement||Mouse IL-2, research grade|