Supporting Online Material


Imaging of Germinal Center Selection Events During Affinity Maturation
Christopher D. C. Allen, Takaharu Okada, H. Lucy Tang, Jason G. Cyster

Supporting Online Material

This supplement contains:
Materials and Methods
SOM Text
Figs. S1 to S9
References
Movies S1 to S12

Corrected (31 January 2007): The legend for movie S11 was revised. The previous legend described a time-lapse sequence not shown in the movie. The corrected legend describes the two sequences that are shown. The originally posted version can be seen here.

This file is in Adobe Acrobat PDF format.

Movie S1
GC B cells are motile and extend dendritic processes. A time-lapse sequence of 18 mum z-projection images shows the dynamics of GC B cells in an axillary lymph node explant 7 d after immunization. White arrowhead, a long process of a GC B cell; yellow arrowhead, cell depicted in Fig. 1B. Elapsed time is shown as hh:mm:ss.

Movie S2
Intravital imaging of GC B cells. A time-lapse sequence of 21 mum z-projection images of a GC in an inguinal lymph node of an anesthetized mouse 7 d after immunization. Cell division and death are annotated in the video. Also indicated is a blood vessel in which four CMTMR-labeled cells (arrowheads) appeared in the image stack within 20 s and disappeared within the next 20 s. Elapsed time is shown as hh:mm:ss.

Movie S3
Plasma cells move with slow velocities and minimal displacements. Time-lapse image sequences of plasma cells in lymph node medullary cords. Left, 36 mum z-projection of the medulla of an axillary lymph node explant in a recipient of VDJ9/κ5 B cells (3% GFP+) and OT-II T cells (approximately 97% CFP+) 7 d after immunization. Right, 30 mum z-projection of the medulla of a mesenteric lymph node explant in a Blimpgfp mouse. Elapsed time is shown as hh:mm:ss.

Movie S4
The boundary between the follicular mantle (FM) and GC. Data are shown as 3-D rotations on the y-axis, and correspond to Fig. 1K. Note that tracks of GC B cells (green) extend into the area occupied by FM B cells (red), and vice versa. The gridlines are separated by 20 mum.

Movie S5
Movement dynamics of cells in dark and light zones. Time-lapse image sequences show cells in the light zone (left panel, 18 mum z-projection) and dark zone (right panel, 33 mum z-projection) from the same GC in a facial lymph node explant 7 d after immunization. FDC in the light zone are labeled with PE immune complexes (IC). An arrowhead highlights a cell that is moving among processes of the FDC network. Elapsed time is shown as hh:mm:ss.

Movie S6
Tracks of cells traveling between light and dark zones. Data are shown as a 3-D rotation on the z-axis for the entire imaging volume analyzed from a GC in a brachial lymph node 7 d after immunization, and correspond to Fig. 2B and movie S8. The PE immune complexes deposited on the FDC network in the light zone are in red, and tracks indicate the path of GFP+ GC B cells that transited from one zone to another. The gridlines are separated by 20 mum.

Movie S7
Cells migrating between dark and light zones. Time-lapse image sequences show maximum intensity projections of the entire 3-D imaging volume analyzed from a GC in a brachial lymph node 7 d after immunization, and correspond to Fig. 2B and movie S7. Tracks of cells moving between dark and light zones are color-coded according to the timescale shown in the bottom-right corner. Gray spheres show the positions of tracked cells over time. An arrowhead highlights a GC B cell moving from the dark zone to the light zone that can easily be followed at this orientation of the imaging volume. The gridlines are separated by 20 mum. Elapsed time is shown as hh:mm:ss.

Movie S8
Most contacts between GC B cells and T cells are transient. A time-lapse sequence of 39 mum z-projection images shows the dynamics of GC B cells and T cells in a brachial lymph node explant 7 d after immunization. In this lymph node, 1-2% of GC B cells were GFP+, and approximately 6% of OT-II T cells were CFP+. Elapsed time is shown as hh:mm:ss.

Movie S9
Stable interactions between cognate antigen-specific B and T cells 36 h after immunization. A time-lapse sequence of 36 mum z-projection images show the dynamics of antigen-engaged B and T cells interacting each other in a facial lymph node. In this lymph node, nearly 100% of VDJ9/κ5 B cells were GFP+ and approximately 97% of OT-II T cells were CFP+. Elapsed time is shown as hh:mm:ss.

Movie S10
Example of a stable interaction between a GC B and T cell. A time-lapse sequence of 6 mum z-projection images shows a migratory conjugate of a GFP+ VDJ9/κ5 B cell and CFP+ OT-II T cell in a GC of an axillary lymph node explant 7 d after immunization. In this lymph node, 1-2% of GC B cells were GFP+, and approximately 97% of OT-II T cells were CFP+. Optimal 6 mum z-projection images capturing the conjugate within a 36 mum z-stack that the conjugate traveled through were used for the image sequence. The time-lapse image sequence is shown twice, first with and then without a migration track of the conjugate. An arrowhead indicates the initial position of the conjugate. Elapsed time is shown as hh:mm:ss.

Movie S11
Fragmentation of GC B cells. Two time-lapse image sequences (30 mum and 54 mum z-projections in the order of appearance) show GFP+ VDJ9/κ5 B cells that were dying and undergoing fragmentation. Fragments of each B cell were taken up by multiple macrophages outlined by dotted lines. Arrowheads indicate dying cells and fragments of dead cells. The last two arrows in the second image sequence indicate fragments that were not taken up by macrophages during imaging. Elapsed time is shown as hh:mm:ss.

Movie S12
GC B cell blebs are carried by GC T cells. Four time-lapse image sequences (45 mum, 39 mum, 36 mum and 24 mum z-projections in the order of appearance) show CFP+ OT-II T cells migrating together with blebs from GFP+ VDJ9/κ5 B cells in GCs. White dots show tracks of the T cell-attached blebs. Elapsed time is shown as hh:mm:ss.

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