OMIP-089
Cattle T-cell phenotyping by an 8-color panel
https://doi.org/10.1002/cyto.a.24718
Eduard O. Roos, William N. Mwangi, Wilhelm Gerner, Ryan Waters, John A. Hammond
Description: This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve), central memory (TCM), effector memory (TEM) and terminal effector (TTE) T cells. Activated cattle T cells (TAV) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve, TAV, TCM, TEM, TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.
Species: Bovine
Tissue: Peripheral blood
Cell Subset: T cell, T memory, Tcrgd
Machine: LSR Fortessa
Panel details
| Marker | Species | Clone | Fluorophore | Purpose | Co-expressions groups | Antigen Density |
|---|---|---|---|---|---|---|
| IgG1 | Bovine | A85-1 | Brilliant Violet 711 | Secondary reagent for CD3 | ||
| CD4 | Bovine | CC8 | T-cell lineage, Helper T cells | T cell | Low | |
| CD8a | Bovine | CC63 | T-cell lineage, Cytotoxic T cells | T cell | Med | |
| FITC | Bovine | polyclonal | Fluorescein | Secondary reagent for CD45RO | T cell | |
| PerCP-Cy5.5 | Bovine | polyclonal | PerCP-Cy5.5 | Secondary reagent for CD62L | T cell | |
| Biotin | Bovine | Streptavidin | Brilliant Violet 421 | Secondary reagent for CD4 | T cell | |
| Live/Dead Fix Near IR (775) | Bovine | Live/Dead Fix Near IR (775) | Viability | |||
| PE-Cy7 | Bovine | Polyclonal | PE-Cy7 | Secondary reagent for TCR gamma/delta | ||
| PE-Texas Red | Bovine | polyclonal | PE-Texas Red | Secondary reagent for CD8a | T cell | |
| PE-Cy5 | Bovine | polyclonal | PE-Cy5 | Secondary reagent for CD25 | T cell | |
| CD62L | Bovine | CC32 | T-cell subset marker, Naïve and Central Memory T cells | T cell | Med | |
| CD25 | Bovine | IL-A111 | Activation marker, Regulatory T cells | T cell | Med | |
| TCR gamma/delta | Bovine | GB21A | T-cell linage marker, γδ T cells | Med | ||
| CD3 | Bovine | MM1A | T-cell lineage | T cell | Med | |
| CD45RO | Bovine | IL-A116 | T-cell subset marker, Memory T cells (Central and Effector) | T cell | High |
