Assay Principle

Markers

 Specificity  Purpose Clone Fluorochrome Laser Filter
 CD45 Leukocyte gating  J33 Krome Orange 405nm 525/40
 CD3 T cell lineage UCHT1 APC-Alexa Flour 750 638 nm 763/43
CD4 T Helper cell lineage  13B8.2 APC 638 nm 660/10
 CD8  Cytotoxic T cell lineage  B9.11  Alexa Fluor 700 638 nm 712/25
 HLA-DR  T cell activation  Immu-357  ECD (PE-Texas Red) 488 nm  610/20
 TCR PAN
α/β		 T cell Receptor Expression
  IP26A PE 488 nm 585/42
 TCR PAN
γ/δ		 T cell Receptor Expression   IMMU510 FITC 488 nm 525/40 
 TCR Vδ1 T cell Receptor Expression   R9.12 PE-Cy7 488 nm 763/43 
 TCR Vδ2  T cell Receptor Expression  IMMU 389 Pacific Blue 488 nm  450/45

Antigen Density

This table provides expression data on the markers included in the panel for the major blood cell types.  This information is a useful aid in panel design and assessing staining results.  Fluorochrome to marker combinations were chosen to reduce spillover between channels where the resolution is needed to identify populations.  The exact resolution achieved will be a property of these choices along with the capabilities of the instrument used for acquisition.

CD3 Measured Antigen Density in peripheral blood

CD4 Measured Antigen Density in peripheral blood

CD8 Measured Antigen Density in peripheral blood

CD45 Measured Antigen Density in peripheral blood

HLA-DR Measured Antigen Density in peripheral blood

TCRab Measured Antigen Density in peripheral blood

TCRgd Measured Antigen Density in peripheral blood

TCRVd1 Measured Antigen Density in peripheral blood

TCRVd2 Measured Antigen Density in peripheral blood

Characterization of Antigen Density. The sample was prepared by staining peripheral blood in a DURAClone IM TCRs Antibody Panel (Part Number B53340) following manufacturer’s instructions and acquired on a CytoFLEX LX N-V-B-Y-R-I Flow Cytometer. Data was analyzed using Kaluza Analysis software.  Dot plots show SSC x marker staining with lymphocytes in green, monocytes in purple and granulocytes in red. Histograms show antigen expression patterns on the major cell types using the same color coding.

Co-expression Patterns and Spillover Rules

DURAClone IM TCR Basic Panel Design Rules

Hierarchical tree structure showing parent-child relationships between the interrogated antigens. Child nodes inherit the properties of the parent nodes above it in the tree. To illustrate, all populations in the analysis will be CD45+, inheriting that property from the parent node. As a principle, spillover from parent to child nodes should be minimized, while spillover from child to parent nodes will not cause a loss of resolution. Nodes at the same hierarchy level without dashed connections are co-expressed antigens whose resolution is impacted by spillover. Colored boxes indicate the expression pattern for the given marker, the lower the expression the more important it will be to avoid spillover from other dyes into that channel. Mutually exclusive antigens in the same level are indicated with a dashed red line. Spillover between these detectors will not cause a loss of resolution as the signals will not be present on the same event. Pairs of antigens that are either double-positive or double-negative are positively correlated antigens that can tolerate spillover, indicated with a dashed gold line.

 

For Research Use Only. Not for use in diagnostic procedures.