Cell Morphology Evaluation Protocol

Cells (3 × 10⁴/per well) were seeded in Lab-Tek™ chamber slides (Thermo-Scientific^®, Waltham, MA, USA) with 400 µL of supplemented medium (Sigma^®, St. Louis, MO, USA) and incubated for 24 h. The treatment duration with IC₅₀ of samples/controls was 24 h. Then, cell fixation began with the removal of the culture medium and the addition of 2% paraformaldehyde (Sigma^®, St. Louis, MO, USA) for 30 min at 37 °C. Morphology changes were evaluated by: i) Hemacolor^® rapid staining (Merck^®, Darmstadt, D.E.) or ii) F-actin staining with Rhodamine-Phalloidin Reagent (Thermo-Fisher™); according to each manufacturer’s instructions. Later, slides were prepared with Entellan^® resin (Merck^®, Darmstadt, D.E.) or Vectashield^®/DAPI mounting medium (Vector Laboratories^®), depending on each assay. Finally, observations were conducted using light microscopy (BX41, Olympus^®, Miami, FL, USA) or confocal microscopy (LSM 700, Zeiss^®, Pleasanton, CA, USA) [30 (link)].