Immunophenotyping of human CD4+ and CD8+ T cell differentiation and exhaustion using flow cytometry (panel A)

MBTP 57  = Miltenyi Biotec-tested panel 57

This application protocol describes the analysis of human CD4+ and CD8+  T cell differentiation and exhaustion markers after T cell stimulation. To demonstrate this state-of-the-art panel for  T cell differentiation as well as T cell exhaustion of both CD4 T cells and CD8 T cells, PBMCs were stimulated for 96 hours using Miltenyi Biotec’s T Cell TransAct™, human. The state of differentiation and exhaustion was defined by CD279 (PD-1), CD336, CD197 (CCR7), and CD45RA expression. 

Protocol

Antibody panel for T cell differentiation and exhaustion

SpecificityClonePurposeFluorochromeDetection filter (nm) (laser)
7-AAD* –Viability –655–730 (blue)
CD279 (PD-1)REA1165T cell differentiation and exhaustionVio® Bright B515525/50 (blue)
CD4*REA623Helper T cellsVioGreen™525/50 (violet)
CD8*REA734Cytotoxic T cellsAPC-Vio 770750 LP (red)
CD45RAREA562T cell differentiation and exhaustionAPC655–730 (red)
CD3REA613T cellsVioBlue®450/50 (violet)
CD366REA635T cell differentiation and exhaustionPE585/40 (blue)
CD197 (CCR7)REA108T cell differentiation and exhaustionPE-Vio 770750 LP (blue)

* Alternative CE-IVD/ASR reagents available. 

Gating strategy

Figure 1: Gating strategy showing the identification of viable, single CD4+ and CD8+ T cells in human PBMCs.  
Cells were gated in an FSC-H and FSC-A dot plot to eliminate doublets (A). Lymphocytes were selected by a SSClow gate which also excluded debris (B). Dead cells were excluded by gating on 7-AAD− cells (C). CD4+ T cells were identified by expression of CD3 and CD4 (D). CD8+ T cells were identified by expression of CD3 and CD8 (E). 
 

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