Figure 6
Figure 6. B cells exit the LN follicle into adjacent lymphatics or into the PCCs. (A) Lymphatics outlined by fluorescent nanodots in the lymph. B cells (green) adoptively transferred the day before imaging. Fluorescent nanodots injected subcutaneously near the tail of the recipient 3 minutes before image (single slice) was acquired by TP-LSM. (B) Percentage of transferred B cells in the lymph, PCCs, and LN follicle. The results from the analyses of 4 separate experiments and shown as percentage of total cells in the follicle, PPC, or in the lymph. The image depth from the capsule was approximately 150 μm. (C) Localization of B cells in the lymph. Shown is a TP-LSM image of transferred B cells (green) in the lymph. (D) Polarity index of B cells in the lymph and the PCCs. Polarity index is calculated by dividing the long axis by the short axis of each cell. Individual cells shown. (E) Tracking B cells in the lymph, LN follicle, and PCCs. TP-LSM was used to track and measure the velocity of B cells in the LN follicle (yellow tracks), PCCs (red tracks), and in the lymph (white) of an inguinal LN. The tracks are shown superimposed over an image and individual velocities plotted below. (F) Location of B cells after blocking entrance or blocking entrance and egress. Left images are from TP-LSM of B cells (green, transferred 42 hours earlier) in the LN of a mouse that received CD62L antibody 24 hours after the cell transfer. Lymphatics (red) revealed by injecting labeled LYVE-1 antibody subcutaneously 12 hours before imaging. In the right 2 images the experimental conditions were similar however FTY720 was injected 18 hours after the cell transfer. Bottom images are from outlined regions in the top images. (G) LN B cells exiting the LN follicle into a PCC. A single image is shown from TP-LSM of the inguinal LN that captures 2 B cells (green) exiting into a PCC. LYVE-1 antibody used to reveal the lymphatics. (H) Transferred B cells in a major LN efferent lymphatic. Adoptively transferred B cells (green) flowing in one of the major efferent lymphatics. Collagen is blue. The pair-wise comparisons of indicated columns were performed using Mann-Whitney test. Scale bars are 100 μm.

B cells exit the LN follicle into adjacent lymphatics or into the PCCs. (A) Lymphatics outlined by fluorescent nanodots in the lymph. B cells (green) adoptively transferred the day before imaging. Fluorescent nanodots injected subcutaneously near the tail of the recipient 3 minutes before image (single slice) was acquired by TP-LSM. (B) Percentage of transferred B cells in the lymph, PCCs, and LN follicle. The results from the analyses of 4 separate experiments and shown as percentage of total cells in the follicle, PPC, or in the lymph. The image depth from the capsule was approximately 150 μm. (C) Localization of B cells in the lymph. Shown is a TP-LSM image of transferred B cells (green) in the lymph. (D) Polarity index of B cells in the lymph and the PCCs. Polarity index is calculated by dividing the long axis by the short axis of each cell. Individual cells shown. (E) Tracking B cells in the lymph, LN follicle, and PCCs. TP-LSM was used to track and measure the velocity of B cells in the LN follicle (yellow tracks), PCCs (red tracks), and in the lymph (white) of an inguinal LN. The tracks are shown superimposed over an image and individual velocities plotted below. (F) Location of B cells after blocking entrance or blocking entrance and egress. Left images are from TP-LSM of B cells (green, transferred 42 hours earlier) in the LN of a mouse that received CD62L antibody 24 hours after the cell transfer. Lymphatics (red) revealed by injecting labeled LYVE-1 antibody subcutaneously 12 hours before imaging. In the right 2 images the experimental conditions were similar however FTY720 was injected 18 hours after the cell transfer. Bottom images are from outlined regions in the top images. (G) LN B cells exiting the LN follicle into a PCC. A single image is shown from TP-LSM of the inguinal LN that captures 2 B cells (green) exiting into a PCC. LYVE-1 antibody used to reveal the lymphatics. (H) Transferred B cells in a major LN efferent lymphatic. Adoptively transferred B cells (green) flowing in one of the major efferent lymphatics. Collagen is blue. The pair-wise comparisons of indicated columns were performed using Mann-Whitney test. Scale bars are 100 μm.

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