Effect of transfected PA317 cell supernatants on human marrow cultures propagated in vitro. Approximately 2 × 106 nucleated marrow cells were plated in D-MEM with heat-inactivated 20% FBS. Supernatants (1 mL) from PA317 cells transfected with SLX-CMV bearing expression cassettes for Vpr mutants, as indicated in the figure, were applied. (A) The following day, cells were washed with media and 200 μL of latex beads (Sigma; catalogue no. L-1398) were added directly to cultures replenished with media, and cultures were incubated at 37°C for an additional 30 minutes, washed with PBS, and photographed. Dark rounded cells are phagocytes ingesting deep-blue latex spheres. (B) More densely seeded cultures retaining large numbers of nonadherent cells were treated with PA317 supernatants as described above, except that no further additions were made. After 24 hours, cells were fixed with 100% methanol and photographed. The effects shown are typical of at least three independent experiments.
