Figure 7. iNKT cell motility during... | Rockefeller University Press

Figure 7.

$Figure 7. iNKT cell motility during innate activation. (A) Fluorescently labeled iNKT cells from a clonal line were incubated with monocyte-derived DCs on ICAM-1–Fc-coated slides in the presence of medium alone or medium containing IL-12 and IL-18 or after the DCs had been pulsed with the strong TCR agonist α-GalCer, and microscopic images were taken once every 15 s. Single cell tracking analysis was performed using ImageJ software. The panels each show the movement traces of seven representative iNKT cell events over a period of 15 min, with the starting point for each event normalized to the center of the picture. Similar results were obtained in three independent experiments using two different iNKT cell clones. (B) The mean velocity for 20–30 different iNKT cells in each condition was calculated by dividing the distance traveled by the time. Horizontal bars within the datasets show the means; p-values shown at the top were calculated by one-way analysis of variance followed by an unpaired Student’s t test. Similar results were obtained in three independent experiments using two different iNKT cell clones. (C) Flow cytometric analysis of tightly adhered iNKT:DC conjugates. Fluorescently labeled iNKT cells from a clonal line and monocyte-derived DCs were coincubated for 24 h and then subjected to vigorous vortexing followed by flow cytometric analysis to determine the fraction of iNKT cells that had remained conjugated to the DCs. iNKT cells and untreated DCs were cultured in medium alone (medium) or in medium containing IL-12 and IL-18 (IL-12&IL-18), or iNKT cells were cultured with LPS-treated or α-GalCer–pulsed DCs as indicated. Each bar represents the mean conjugate frequency from three independent analyses, with error bars showing the standard deviations of the means. (D) Microscopic analysis of iNKT cells from a clonal line labeled with CFSE (green) incubated for 2 h with DCs labeled with DiD (red) under conditions corresponding to those described in C. Similar results were obtained in three independent experiments using two different iNKT cell clones.$

iNKT cell motility during innate activation. (A) Fluorescently labeled iNKT cells from a clonal line were incubated with monocyte-derived DCs on ICAM-1–Fc-coated slides in the presence of medium alone or medium containing IL-12 and IL-18 or after the DCs had been pulsed with the strong TCR agonist α-GalCer, and microscopic images were taken once every 15 s. Single cell tracking analysis was performed using ImageJ software. The panels each show the movement traces of seven representative iNKT cell events over a period of 15 min, with the starting point for each event normalized to the center of the picture. Similar results were obtained in three independent experiments using two different iNKT cell clones. (B) The mean velocity for 20–30 different iNKT cells in each condition was calculated by dividing the distance traveled by the time. Horizontal bars within the datasets show the means; p-values shown at the top were calculated by one-way analysis of variance followed by an unpaired Student’s t test. Similar results were obtained in three independent experiments using two different iNKT cell clones. (C) Flow cytometric analysis of tightly adhered iNKT:DC conjugates. Fluorescently labeled iNKT cells from a clonal line and monocyte-derived DCs were coincubated for 24 h and then subjected to vigorous vortexing followed by flow cytometric analysis to determine the fraction of iNKT cells that had remained conjugated to the DCs. iNKT cells and untreated DCs were cultured in medium alone (medium) or in medium containing IL-12 and IL-18 (IL-12&IL-18), or iNKT cells were cultured with LPS-treated or α-GalCer–pulsed DCs as indicated. Each bar represents the mean conjugate frequency from three independent analyses, with error bars showing the standard deviations of the means. (D) Microscopic analysis of iNKT cells from a clonal line labeled with CFSE (green) incubated for 2 h with DCs labeled with DiD (red) under conditions corresponding to those described in C. Similar results were obtained in three independent experiments using two different iNKT cell clones.