SAP^– iNKT cells accumulate actin but demonstrate impaired MTOC polarization at the IS. (A) Conjugates of murine SAP⁺ or SAP^– iNKT cells and antigen-loaded EL4 cells were prepared, fixed at 4 hours, stained, and evaluated by transmitted light (TL) and spinning disk confocal microscopy (Zeiss Observer-Z.1 with Hamamatsu cooled-CCD camera) by using fluorescently labeled phalloidin to detect F-actin. The magnification of the objective lens was ×63 and the numerical aperture was 1.45. Scale bar: 5 μm. (B) Graph represents mean percentage ± SEM of conjugates that were positive (⁺ve) for F-actin accumulation at the IS. Data are pooled from 3 experiments; scoring was for ≥20 conjugates per condition. Conjugates were evaluated in a blinded fashion and scored positive for F-actin accumulation at the synapse when there was enhanced staining at the site of iNKT-target-cell interface. (C-G) Conjugates were similarly evaluated and scored in a blinded fashion for MTOC polarization at the synapse. (C) Conjugates of SAP⁺ iNKT cells and unloaded EL4 cells were analyzed for localization of the MTOC at the IS. (D-E) Representative conjugates of SAP⁺ or SAP^– iNKT cells and antigen-loaded EL4 cells analyzed at 4 or 14 hours are shown. White arrowheads indicate the centrosome. (F) MTOC polarization at the IS in SAP⁺ and SAP^– NKT cells was assessed by measuring its distance from the IS at 4 and 14 hours. (G) Average percentage of conjugates positive (⁺ve) for MTOC at the synapse. Each mean represents data from at least 25 conjugates ± SEM. Statistical significance was determined by unpaired two-tailed t test. **P < .001.