Figure 5.
Figure 5. TLR4 ligand–treated B cells enter ongoing germinal center reactions and focus in the dark zone. (A) Germinal center imaging. MIPs of transferred TLR4-stimulated B cells (transferred 2 d before imaging) in iLN of mice immunized 1 wk previously with HEL. The top left shows a cluster of the TLR4-stimulated B cells (arrow) 1 wk after immunization. Also shown are images of TLR4-stimulated B cells in the light zone region, at the interface dark zone–light zone region, and in the dark zone region of the germinal center (bottom). Location of the cells defined by FDC-M2–positive cells were revealed by Alexa Fluor 488–labeled FDC-M2 injected 1 d before imaging. Bars, 50 µm. (B) Tracking. TLR4-stimulated B cells within the germinal center light zone. TLR4-stimulated cells imaged intravitally (FDC-M2, green; TLR4-stimulated B cell, red) were tracked for 20 min. Tracks (white) are from those cells that remained in the imaging space for at least 10 min. Bar, 50 µm. (C) Dark zone interactions. A single slice is shown from intravital TP-LSM showing the location of TLR4-stimulated B cells in the dark zone region. The centers of each cell are indicated with a gray ball. Three pairs of interacting B cells are indicated with white arrows. In one pair the centers were superimposed. Bar, 20 µm. (D) Motility parameters. Cell motilities in light zone (LZ), in dark zone (DZ), and outside of the germinal center (Non-GC) were determined. Each data point represents a single cell and blue, red, and yellow bars indicate mean values (**, P < 0.05). (E) Tracks. Shown are superimposed tracks of TLR4-stimulated cells of each indicated type in the x, y plane setting the starting coordinates to the origin. Tracks of 30 cells in the indicated sites tracked for 15 min of a 20-min imaging experiment. (F) Velocity profiles. Magnitudes are shown of the instantaneous velocities of TLR4-stimulated B cells in the light zone and the dark zone. An interaction with another cell is indicated by shading. Experiments were performed five times using multiple recipient mice.

TLR4 ligand–treated B cells enter ongoing germinal center reactions and focus in the dark zone. (A) Germinal center imaging. MIPs of transferred TLR4-stimulated B cells (transferred 2 d before imaging) in iLN of mice immunized 1 wk previously with HEL. The top left shows a cluster of the TLR4-stimulated B cells (arrow) 1 wk after immunization. Also shown are images of TLR4-stimulated B cells in the light zone region, at the interface dark zone–light zone region, and in the dark zone region of the germinal center (bottom). Location of the cells defined by FDC-M2–positive cells were revealed by Alexa Fluor 488–labeled FDC-M2 injected 1 d before imaging. Bars, 50 µm. (B) Tracking. TLR4-stimulated B cells within the germinal center light zone. TLR4-stimulated cells imaged intravitally (FDC-M2, green; TLR4-stimulated B cell, red) were tracked for 20 min. Tracks (white) are from those cells that remained in the imaging space for at least 10 min. Bar, 50 µm. (C) Dark zone interactions. A single slice is shown from intravital TP-LSM showing the location of TLR4-stimulated B cells in the dark zone region. The centers of each cell are indicated with a gray ball. Three pairs of interacting B cells are indicated with white arrows. In one pair the centers were superimposed. Bar, 20 µm. (D) Motility parameters. Cell motilities in light zone (LZ), in dark zone (DZ), and outside of the germinal center (Non-GC) were determined. Each data point represents a single cell and blue, red, and yellow bars indicate mean values (**, P < 0.05). (E) Tracks. Shown are superimposed tracks of TLR4-stimulated cells of each indicated type in the x, y plane setting the starting coordinates to the origin. Tracks of 30 cells in the indicated sites tracked for 15 min of a 20-min imaging experiment. (F) Velocity profiles. Magnitudes are shown of the instantaneous velocities of TLR4-stimulated B cells in the light zone and the dark zone. An interaction with another cell is indicated by shading. Experiments were performed five times using multiple recipient mice.

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