Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells
Transcrição
γδ T cells express T cell receptors, TCRs, comprising gamma and delta chains with variable and constant domains.
CD4+ Vδ1+ T cells — a γδ T cell subpopulation — co-express the variable Vδ1 chain and the CD4 co-receptor. These cells migrate into inflamed tissues and transdifferentiate into functionally mature CD4+ or CD8+ αβ T cells — mediating an immune response.
To isolate CD4+ Vδ1+ T cells from human peripheral blood mononuclear cells, PBMCs, resuspend the cells in a buffer. Use an Fc-blocking reagent to block the cell-surface Fc receptors and prevent non-specific antibody binding.
Add anti-Vδ1 antibodies conjugated with the fluorophore FITC, which bind to the γδ T cells' Vδ1 antigens. Incubate with anti-FITC antibody-coupled magnetic microbeads, which attach to the FITC dye on the Vδ1 T cell-bound antibodies.
Pass the mixture through a magnetic column to attract the bead-bound Vδ1+ T cells; the Vδ1- T cells remain unbound and are subsequently flushed out. Forcefully flush the column by pushing the buffer through it to detach the Vδ1+ T cells.
Incubate the cells with anti-CD4 antibody-coated magnetic beads, which bind to the cells' CD4 co-receptor. Place the tube in a magnet to attract the bead-bound CD4+ Vδ1+ T cells; the CD4- Vδ1+ T cells remain unbound.
Resuspend the CD4+ Vδ1+ T cells in a medium. Add a bead-detaching reagent to release the beads from the cells. Replace the tube on the magnet to separate the beads.
Collect the CD4+ Vδ1+ cell supernatant for further analysis.
