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. 2021 Mar 22;10:e56020. doi: 10.7554/eLife.56020

Figure 3. YellowCaB cells form productive germinal centers in vivo and show active B cell receptor signaling after cell-to-cell contacts.

(a) Histological analysis of host mouse lymph nodes after adoptive transfer of YellowCaB cells. TN-XXL (green)-positive cells cluster in IgD (blue)-negative regions; a germinal center phenotype is confirmed by PNA staining (red). Scale bar 50 µm. (b) Stills of ratiometric intravital imaging of adoptively transferred YellowCaB cells. 3D surface rendering and single-cell tracking (track line in yellow) with relative color coding ranging from blue = low ΔR/R to red = high ΔR/R (c) Histogram showing segmented objects binned due to colocalization intensity within bin width of 20 AU and biexponential fit of data. Total number of objects = 6869. A curve decay of <10% was set as threshold, parting transient from strong B cell–FDC contact. All cells with colocalization intensity <1 were assigned negative. (d) Colocalization intensities of tracked cells 1 and 2 over time versus Förster resonance energy transfer signal change of cells 1 and 2 over time. Contact events to FDCs were assigned numbers #1, #2, and #3.

Figure 3.

Figure 3—figure supplement 1. Cell velocity versus calcium flux.

Figure 3—figure supplement 1.

Intravital imaging parameters of germinal center YellowCaB cell making multiple contacts with FDCs (#1–3, red dashed lines). Top: ΔR/R Förster resonance energy transfer signal change of tracked cell over time; the first five time points were taken as baseline. Middle: instantaneous velocity over time. Bottom: first derivative of displacement rate (DR) in direction of X (orange), Y (green), and Z (blue). All DR′=0 means complete halt of cell dislocation.