Lab tek 2 chamber slide

The In Situ Cell Death Detection kit(Roche Diagnostics, Basel, Switzerland) was used to detect apoptosis in the cell culture. HCT 116 cells were seeded at 3000 cells/well onto Lab-Tek II Chamber Slides (Nalge Nunc International, Tokyo, Japan) and incubated overnight. Cells were incubated with linalool (0, 100 μmol/L) or 50 nM staurosporine as a positive control for 24 h. Cells were incubated with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) reaction mixture according to the manufacturer’s instructions.
A flow cytometer (Muse Cell Analyzer) and Muse Annexin V & Dead Cell Kit (Merck Millipore, Darmstadt, Germany) were used for quantitative analyses of live, early and late apoptosis, and cell death in suspension cell lines. HCT 116 cells were seeded at a density of 1 × 10⁵ cells per dish (diameter, 6 cm) and incubated overnight. Cells were incubated with different concentrations of linalool (0, 10, 100, 1000 μmol/L) and 50 nM staurosporine as the positive control for 24 h. Cells were prepared with Annexin V and Dead Cell reagent according to the manufacturer’s instructions, and the samples were evaluated using the MUSE analyzer with the appropriate thresholds for cell size and apoptotic profile.

Cultured cells were plated onto Lab-Tek II chamber slides (Nalge Nunc International, Rochester, NY, USA). The cells were washed twice with phosphate-buffered saline (PBS) (-), fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 in PBS (-) for 3 min at room temperature. Non-specific binding was blocked with CAS-Block (Invitrogen, Carlsbad, CA, USA) for 7 min at room temperature before the primary antibody reaction. Then, cells were incubated with rabbit anti-KIF11 antibody (catalog no. HPA010568; Sigma-Aldrich) in PBS (-) containing 1% bovine serum albumin (BSA) for 60 min at room temperature in a wet box. After washing with PBS (-), the cells were stained with Alexa 488-conjugated anti-rabbit secondary antibody (Life Technologies, Grand Island, NY, USA) for 60 min at room temperature in a wet box to protect from light. After washing again with PBS (-), the cells were mounted using Vectashields mounting medium containing DAPI (Vector Laboratories, Inc. Burlingame, CA, USA) and were visualized using BZ-X710 (Keyence, Osaka, Japan).