Light optical microscope

Cell proliferation was determined using the SRB assay^{46 (link)}. Briefly, cells were seeded in 96-well plates at 5.0 × 10³ (for HCT116 and HT-29) and 7.5 × 10³ (for SW-837) cells/well and 24 h later treated with PMA, Roy-Bz, or vehicle for 48 h. IC₅₀ (concentration that causes 50% of growth inhibition) values were determined from the concentration–response curves. The percentage of viable cells was determined using the Trypan Blue Assay. Briefly, HCT116 cells were seeded in 24-well plates at 6 × 10⁴ cells/well for 24 h, followed by treatment with Roy-Bz for 24 h. Cells were then harvested and resuspended in 0.4% Trypan Blue (Sigma-Aldrich), and the number of viable/dead cells were counted using a Leica light optical microscope (Wetzlar). Vehicle (0.25% DMSO) was included as a control.

Genotoxicity was analyzed by the cytokinesis‐block micronucleus assay in human lymphocytes, as described (Soares et al., 2016). Briefly, fresh peripheral blood samples were collected from healthy volunteers into heparinized vacutainers. Blood samples, suspended in RPMI medium supplemented with 10% FBS, were treated with 7, 14, and 21 μm of DIMP53‐1, 1 μg·mL⁻¹ cyclophosphamide (positive control), or solvent for 44 h. Cells were thereafter treated with 3 μg·mL⁻¹ cytochalasin B (cytokinesis preventive) for 28 h. Lymphocytes were isolated by density gradient separation (Histopaque‐1077 and ‐1119), fixed in 3 : 1 methanol/glacial acetic acid, and stained with Wright stain. For each sample, 1000 binucleated lymphocytes were blindly scored using a Leica light optical microscope (Wetzlar, Germany); the number of micronuclei per 1000 binucleated lymphocytes was recorded.

Genotoxicity was analyzed by cytokinesis-block micronucleus assay in lymphocytes as described [34 (link)]. Fresh peripheral blood samples were collected from healthy volunteers into heparinized vacutainers. Blood samples, suspended in RPMI medium supplemented with 10% FBS, were treated with the GI₅₀ concentration of SLMP53-1, DMSO only or 1 μg/mL cyclophosphamide (known mutagenic agent; positive control; Sigma-Aldrich) for 44 h. Cells were thereafter treated with 3 μg/mL cytochalasin B (cytokinesis preventive; Sigma-Aldrich) for 28 h. Lymphocytes were isolated by density gradient separation (Histopaque-1077 and -1119; Sigma-Aldrich,), fixed in 3:1 methanol/glacial acetic acid, and stained with Wright stain (Sigma-Aldrich). For each sample, 1000 binucleated lymphocytes were blindly scored using a Leica light optical microscope (Wetzlar); the number of micronuclei per 1000 binucleated lymphocytes was recorded.