Phagocytosis and Macropinocytosis

To measure efficiency of phagocytosis, 3 × 10⁵ D. discoideum cells were washed once, resuspended in either 1 mL of HL5 or 1 mL of phosphate buffer (PB: 2 mM Na₂HPO₄, 14.7 mM KH₂PO₄, pH 6.5) containing 1 μl of FITC polystyrene beads (Fluoresbrite plain YG 1 micron, Polysciences), and incubated for 30 min in a shaken suspension. To assess macropinocytosis, cells were incubated in either HL5 or PB containing 10 μg/mL Alexa-647 Dextran (Life Technologies) for 30 min. Then, cells were washed in ice cold HL5 supplemented with 0.1% NaN₃ and internalized fluorescence was measured by flow cytometry. Ingested fluorescence was determined for each strain and normalized to internalization in WT cells.

For phagocytosis kinetic measurements, cells were incubated in 1 mL of HL5 containing 1 μL FITC polystyrene beads, and a 100 μL aliquot of the suspension was taken at each indicated time point. Cells were then washed, and phagocytosis was analyzed as described above.

When mixed populations containing GFP-expressing cells (FL1 channel) were used, they were allowed to ingest red fluorescent beads (Fluoresbrite Polychromatic Red, Polysciences, FL2 channel). The fluorescence values were corrected to compensate for leakage of fluorescence between the FL1 and FL2 channels. To ascertain that the cells were adequately identified as GFP-positive or -negative, control populations were analyzed. When lrrkA KO cells were mixed with WT-GFP cells (Figure 2), lrrkA KO and WT-GFP cells were analyzed separately in parallel, and each cell type was present almost exclusively in the assigned window (99.9 and 99.5%, respectively). Similarly, when lrrkA KO cells were transfected with a GFP-LrrkA expressing plasmid, untransfected cells were analyzed in parallel and were almost exclusively (99.8%) found in the GFP-negative window. In the transfected population, GFP-negative cells represented 24% of the total in a control sample (no beads phagocytosed), and 23.6% in the sample of cells having ingested beads, indicating that no significant number of cells were assigned to the wrong population. In all measures of phagocytosis, median values were used, ensuring that a small percentage of cells improperly identified as GFP-negative or GFP-positive would not significantly affect the results.

To produce conditioned cellular supernatant, D. discoideum cells were grown to a density of 4 × 10⁶ cells in a shaken suspension in HL5 medium. The cell supernatant was recovered by centrifuging cells at 1,500 g for 10 min. To assess the effect of conditioned medium on phagocytosis, cells were incubated for 4 h in fresh or conditioned medium in six-well plates (450,000 cells per well in 1.5 mL), then resuspended, and allowed to phagocytose beads for 5 min in fresh HL5 medium.