Labtak chamber slides

Confocal analyses were performed in Olympus FluoView FV1000 MPE microscope that is equipped with multiphoton capabilities (MaiTai DSBB-OL, 710–990 nm; MaiTai DSHP-OL, 690–1,040 nm). NK cells were plated in poly-l-lysine-coated (MilliporeSigma) LabTak chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) for staining and imaging. Cells were fixed/permeabilized with 4% paraformaldehyde for 15 min and blocked for 1 h with 3% BSA and 0.1% Triton X-100 in sterile PBS. Staining with anti-tubulin (Cell Signaling Technologies) was performed overnight at 4°C. Cells were stained with fluorescent phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) or DAPI for 30 and 5 min, respectively. The following dilutions of reagents were used: anti-tubulin: 1:200, Phalloidin-Red (F-actin): 1:1,000, and DAPI: 1:1,000. Alexa Fluor-conjugated secondary antibodies were used at a 1:2,000 dilutions. Stained slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), and images were taken with a 20× or 40× lenses. Data were analyzed and fluorescent intensities were calculated using Olympus software.

BE(2)-C, CHP134 and Kelly cells were seeded into Lab-Tak™ Chamber slides (Thermo Scientific, Pittsburgh, PA), and treated with vehicle control, 500nM, 8nM vincristine, or combination for 24 hours. Cells were then fixed for 15 minutes in 4% paraformaldehyde, blocked with 2% horse serum, and incubated with mouse anti-α-tubulin antibody (Sigma, Ct Louis, MO), Alexa Fluor 594-conjugated goat anti-mouse antibody (Thermo Scientific) and DAPI (Vector Laboratories, Burlingame, CA, USA). Cell images were captured under a fluorescence microscope (Zeiss, Oberkochen, Germany), and cells with aberrant mitotic spindles were quantified.