Hemacolor stain

Cell fusion was further confirmed by evaluating the presence of syncytium in transfected B16 cells. Briefly, B16 cells seeded on coverslips at a 40-60% of confluence were transfected with 0.5 μg of the pIRES-ARV plasmid using Lipofectamine 2000 (Invitrogen, 11668027) according to the manufacturer's recommendations and 2.5 μg of the same plasmid present in chitosan NPs synthesized at an N/P 20. At 48 hours posttransfection, the cells were washed with PBS and incubated with the CellMask Orange plasma membrane stain probe (Life Technologies, C10045) according to the manufacturer's recommendations. After fixation with 3.75% paraformaldehyde for 30 min at 37°C, the cells were incubated with DAPI 0.5 mg/ml for 5 min. Covers were mounted on slides with DABCO and observed using the Zeiss LSM 800 confocal microscope (Universidad de Santiago de Chile). For hemacolor stain, cells were washed and fixed with at 3 : 1 solution of methanol : acetic acid for 15 min and stained with hemacolor stain (Sigma-Aldrich) according to the manufacturer. Syncytia were counted in 5 random planes using a conventional inverted microscope.

The assay was performed according to Stoker et al (1987) (link). MDCK cells were seeded at low density (2,000 cells/well on a 12-well plate, #353043 Falcon; Corning) in DMEM supplemented with 10% FCS to form compact colonies. After treatment, colony dispersion was observed, and the cells were fixed and coloured by the Hemacolor stain (Merck) according to the manufacturer’s instructions. Representative images were captured using a phase-contrast microscope with 40× and 100× magnification (Nikon Eclipse TS100).