MBTP 19: Immunophenotyping of regulatory T cells from mouse spleen using flow cytometry

Miltenyi Biotec-tested panel 19 (MBTP 19)

This application protocol describes the flow cytometric analysis of regulatory T cells (Tregs) after spleen dissociation from healthy C57BL/6 mice. Mouse spleens are easily dissociated using the gentleMACS™ Dissociators to quickly obtain viable single cell suspensions ready for flow cytometric analysis.


Gating strategy showing the analysis of regulatory T cells (Tregs) from mouse spleen. Splenocytes from C57BL/6 mice were stained using the previously described Treg panel to analyze regulatory T cells. Samples were initially gated on single cells using FSC-H/FSC-A gating and on live cells using Viobility-negative gating (data not shown). Tregs represent a small population of CD3+ CD4+ T cells (A), that characteristically expresses high levels of CD25 (B, C).  CD3+ CD4+ CD25+ T cells express the transcription factor forkhead/winged-helix transcriptional regulator (FoxP3) (G). Moreover, CD3+ CD4+ T cells can be directly gated on FoxP3 (D) and CD357 (E). A subpopulation of CD4+ FoxP3+ Tregs also expresses CD134 (F).


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