Figure 6. Externally-triggered egress in vitro.

Egress was assessed in co-cultures of YFP-RH-infected thioglycolate-elicited macrophages (PEM) with washed exudate cells obtained from mice infected for 5d with wild-type RH. (A, B) YFP fluorescence is displayed from timelapse images (10 min intervals) of adherent PEM exposed at 0 h to either blank medium (A) or a 10-fold excess of exudate cells (B). Egress events that had occurred by 2h are indicated by arrows (lower panels). Scale bar: 20 μm. The quantitation of these egress events (C) represents the mean ± S.E. from either 3 (exudate) or 8 (medium) fields. The data are representative of two similar experiments. (D) The time distribution of exudate cell-induced egress events from the same experiment is displayed as a histogram. The settling of most exudate cells onto the PEM monolayer requires 15 - 20 min. (E) Flow cytometric assay of externally-triggered egress (ETE). Donor PEM infected overnight with YFP-RH (moi=0.2) were labeled with DDAO-SE and mixed (1:9) with PEM similarly infected with wild-type RH. Basal egress was then monitored by incubation of this mixture (None) for 1 h in non-adherent dishes, followed by determination of the percent YFP-RH not donor cell-associated (NDAP). ETE was measured as the additional NDAP generated by the addition of a 5-fold excess of washed exudate cells (Exudate). Control incubations were performed in the presence of a similar excess of uninfected or wild-type RH-infected PEM. ** p < 0.01 (n = 4). The data are representative of two similar experiments.