DNA staining, flow cytometry, and confocal microscopy

Log-phase promastigotes were sedimented and resuspended in 1 mL PBS and fixed in 0.4% paraformaldehyde for 1 min at room temperature. Cells were sedimented, resuspended in 100 mL PBS and permeabilized with 1 mL methanol for 15 min on ice. Next, cells were sedimented, resuspended in 1 mL PBS and incubated for 10 min at room temperature. Staining was performed by adding 500 mL of PI-RNase mix (13 mg/mL each) to the cell suspension and incubating for 1 h at room temperature. Labeled cells were washed once in PBS and analyzed on a FacsCANTO II (BD Biosci-ences) flow cytometer. Hybrids ploidy was inferred by comparison with the DNA content of the parental lines.