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Analysis of human thymocyte development with a 14-color flow cytometry panel

Sarah-Jolan Bremer, Laura Glau, Christina Gehbauer, Annika Boxnick, Daniel Biermann, Jörg Siegmar Sachweh, Eva Tolosa, Anna Gieras Cytometry PART A, Volume 99, Issue 9, 875-879 (2021)

PURPOSE: This panel was designed for the identification and detailed characterization of the different developmental steps of human thymocytes. We optimized the panel for fresh tissue in order to provide an unbiased analysis of T cell development. Accurate selection of antibodies and precise gating allow us to phenotype 14 major stages of human thymocyte development and illustrate the trajectories of T cell development from early thymic progenitors (ETP) to mature T cells that are ready to populate the periphery. The panel identifies ETPs, T-lineage-committed cells (TC), CD34-positive immature single-positive CD4 cells (ISP4 CD34+), CD34-negative immature single-positive CD4 cells (ISP4 CD34-), CD45-low early double-positive cells (EDP CD45low), CD45-high early double-positive cells (EDP CD45high), late double-positive cells (LDP), single-positive CD4 cells (SP4), single-positive CD8 cells (SP8), ready-to-egress single-positive CD4 cells (rSP4), ready-to-egress single-positive CD8 cells (rSP8), T γδ cells (Tγδ), T regulatory cells (Treg), and ready-to-egress T regulatory cells (rTreg). To highlight important checkpoints during T cell development, we added antibodies relevant for specific developmental steps to the panel. These include CD1a to define TCs, CD28 as a marker for ß-selection and CD69 in combination with CD45RA to determine the maturation stage of thymocytes shortly before they become ready to egress the thymus and colonize the periphery. Moreover, Annexin V, as a marker for apoptosis, provides valuable extra information concerning the apoptotic death of thymocytes. Currently, we use this panel to identify aberrations in T cell development in health and disease.

CELL TYPE: Human thymocytes


Marker Target Clone Fluorochrome Purpose
CD25 Human BC96 Brilliant Violet 421 T regulatory cells
CD45 Human HI30 Brilliant Violet 510 Leucocytes
TCR gamma/delta Human 11F2 Brilliant Violet 605 T γδ cells
CD3 Human OKT3 Brilliant Violet 650 T cells
CD45RA Human HI100 Brilliant Violet 711 Lineage marker during early and late developmental stages
CD8a Human RPA-T8 Brilliant Violet 785 CD8 T cells and double-positive cells
CD1a Human HI149 FITC T cell lineage commitment
CD69 Human FN50 PerCP-Cy5.5 Tissue retention marker
CD34 Human 563 PE Hematopoietic stem and progenitor cells
CD4 Human RPA-T4 PE-Dazzle 594 CD4 T cells, ISP4 and double-positive cells
CD28 Human CD28.2 PE-Cy7 ß-selection
Annexin V All Species Alexa Fluor 647 Apoptosis
CD7 Human M-T701 Alexa Fluor 700 T cell lineage
Dead Cell All Species Alexa Fluor 750 viability