Fig. 7.

Analysis of LPS B-cell directional motion toward CpG mOVA B-cell–OTII T-cell pairs. (A) 3D directional persistence of cell movement relative to the origin (red dot) is assessed using the equation (D2 − D1 + D3 − D2)/(d2 + d3) where D = x, y, z displacement relative to the origin and d = x, y, z distance per step. This method of analysis is applicable to any number of sequential steps and generates a single parameter for 3D directional persistence. (B) Diagram of directional persistence value extremes. A value of −1 represents cell movement directly toward the origin, 0 represents neutral movement relative to the origin, and a value of 1 represents 3D movement in a straight line away from the origin. We chose to measure directional persistence over two sequential steps to smooth any single-step tracking anomalies that can occur if a cell does not move a significant distance from the point of origin. (C) Histogram of directional persistence of control WT B cells without treatment imaged in the absence of OTII T cells. Arrow indicates mean value of directional persistence (mean = 0.13 ± 0.01, n = 20 cells; data are from three separate experiments). (D) Directional persistence of LPS B cells tracked for 8 min before contact with CpG B-cell–T-cell pairs. Arrow indicates mean values of directional persistence. Data are from three separate experiments. mean = 0.45 ± 0.02; n = 20 cells. (E) Mean directional persistence values of control B cells from histogram in C (control B); CpG B cells that did not interact with a B–T pair (CpG non-int); CpG B cells that did interact with an LPS B–T pair (CpG int); a swarm of LPS B cells near a CpG B–T pair (swarm B); LPS B cells that did not interact with a B–T pair (LPS non-int); and LPS B cells that interacted with a CpG B–T pair (LPS int).