A new study sheds light on the many roles of the critical protein Themis in Tcell biology—and provides clues that could guide the manipulation of T cells in CellTherapy and other clinical applications. SciSignal
EDTA) for 5 min at room temperature, followed by centrifugation. After centrifugation, the cell pellet was resuspended in 5 ml of single-cell medium. Cell numbers were routinely counted using a NovoCyte flow cytometer from Agilent Technologies.T cells were purified by a negative selection protocol.
and the purity was greater than 95% as determined by flow cytometry. The following antibodies were included in the depletion cocktail: anti-mouse CD4, anti-mouse/human CD45R/B220, anti-mouse CD25, anti-mouse/human CD44, anti-mouse/human CD11b, anti-mouse CD11c, anti-mouse NK-1.1, anti-mouse CD49b , anti-mouse F4/80, anti-mouse Ly-6G/Ly-6C , anti-mouse CD105, anti-mouse TCR γ/δ, and anti-mouse TER-119/erythroid cells.
Flow cytometry was performed on a Fortessa X-20 or Quanteon equipment and analyzed with FlowJo software. For surface marker staining, the following antibodies were used: anti-CD44, anti-CD122, and anti-CD25. Cells were stained in fluorescence-activated cell sorting buffer at 4°C in the dark for 30 min.Naïve WT and cKO OT-I T cells bearing different congenic markers were collected and mixed at 1-to-1 ratio.
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