Figure 1.

Polarization of pre-TCR components in DN3 cells in vitro and in situ. (A and B) DN3 cells were incubated with OP9-DL1 cells for 14 h and fixed and stained for α-tubulin to mark the MTOC and a TCR-associated protein (as shown). Z-stack images were acquired using confocal microscopy, and representative images are shown as maximum projections (A). After triaging for cells in which the MTOC was recruited to the interface with an OP9-DL1 cell, the percentage of cells in which the TCR component was polarized to the interface (white asterisks) with the OP9-DL1 cell was determined by blind scoring (B). The total number of scored conjugates per marker is 75 (25 cells per biological replicate). (C) DN3 cells in a section of an intact thymus were stained for CD25 only as a nonpolarized control. (D and E) DN3 cells were stained for CD25 and either pTα (C) or pLCK³⁹⁴. Images were acquired using widefield fluorescent microscopy (Vectra 3 automated quantitative pathology imaging system), and representative images of polarized (top row) and nonpolarized (bottom row) are shown. Total number of scored cells: n = 156 (pTα) and n = 134 (pLCK³⁹⁴). Scale bars represent 5 µm (A) and 10 µm (C–E). Error bars (B) represent SEM.